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1.  Developmentally regulated expression and complex processing of barley pri-microRNAs 
BMC Genomics  2013;14:34.
MicroRNAs (miRNAs) regulate gene expression via mRNA cleavage or translation inhibition. In spite of barley being a cereal of great economic importance, very little data is available concerning its miRNA biogenesis. There are 69 barley miRNA and 67 pre-miRNA sequences available in the miRBase (release 19). However, no barley pri-miRNA and MIR gene structures have been shown experimentally. In the present paper, we examine the biogenesis of selected barley miRNAs and the developmental regulation of their pri-miRNA processing to learn more about miRNA maturation in barely.
To investigate the organization of barley microRNA genes, nine microRNAs - 156g, 159b, 166n, 168a-5p/168a-3p, 171e, 397b-3p, 1120, and 1126 - were selected. Two of the studied miRNAs originate from one MIR168a-5p/168a-3p gene. The presence of all miRNAs was confirmed using a Northern blot approach. The miRNAs are encoded by genes with diverse organizations, representing mostly independent transcription units with or without introns. The intron-containing miRNA transcripts undergo complex splicing events to generate various spliced isoforms. We identified miRNAs that were encoded within introns of the noncoding genes MIR156g and MIR1126. Interestingly, the intron that encodes miR156g is spliced less efficiently than the intron encoding miR1126 from their specific precursors. miR397b-3p was detected in barley as a most probable functional miRNA, in contrast to rice where it has been identified as a complementary partner miRNA*. In the case of miR168a-5p/168a-3p, we found the generation of stable, mature molecules from both pre-miRNA arms, confirming evolutionary conservation of the stability of both species, as shown in rice and maize. We suggest that miR1120, located within the 3′ UTR of a protein-coding gene and described as a functional miRNA in wheat, may represent a siRNA generated from a mariner-like transposable element.
Seven of the eight barley miRNA genes characterized in this study contain introns with their respective transcripts undergoing developmentally specific processing events prior to the dicing out of pre-miRNA species from their pri-miRNA precursors. The observed tendency to maintain the intron encoding miR156g within the transcript, and preferences in splicing the miR1126-harboring intron, may suggest the existence of specific regulation of the levels of intron-derived miRNAs in barley.
PMCID: PMC3558349  PMID: 23324356
MicroRNA; Pri-microRNA processing; MicroRNA genes; Splicing; Alternative splicing; Introns; Barley
2.  MIR846 and MIR842 comprise a cistronic MIRNA pair that is regulated by abscisic acid by alternative splicing in roots of Arabidopsis 
Plant molecular biology  2013;81(4-5):447-460.
MicroRNAs (miRNAs) are ~21-nucleotide long endogenous small RNAs that regulate gene expression through post-transcriptional or transcriptional gene silencing (PTGS/TGS) and/or translational inhibition. miRNAs can arise from the “exon” of a MIRNA gene, from an intron (e.g. mirtrons in animals), or from the antisense strand of a protein coding gene (natural antisense microRNAs, nat-miRNAs). Here we demonstrate that two functionally related miRNAs, miR842 and miR846, arise from the same transcription unit but from alternate splicing isoforms. miR846 is expressed only from Isoform1 while in Isoforms2 and -3, a part of pre-miR846 containing the miRNA* sequence is included in the intron. The splicing of the intron truncates the pre-MIRNA and disrupts the expression of the mature miR846.. We name this novel phenomenon splicing-regulated miRNA. Abscisic acid (ABA) is shown to mediate the alternative splicing event by reducing the functional Isoform1 and increasing the non-functional Isoform3, thus repressing the expression of miR846 concomitant with accumulation of an ABA-inducible target jacalin At5g28520 mRNA, whose cleavage was shown by modified 5′-RACE. This regulation shows the functional importance of splicing-regulated miRNA and suggests possible mechanisms for altered ABA response phenotypes of miRNA biogenesis mutants. A. lyrata-MIR842 and Aly-MIR846 have conserved genomic arrangements with A. thaliana and candidate target jacalins, similar primary transcript structures and intron processing, and better miRNA-miRNA* pairings, suggesting that the interactions between ABA, MIR842, MIR846 and jacalins are similar in A. lyrata. Together, splicing-regulated miRNAs, nat-miRNAs/inc-miRNAs and mirtrons illustrate the complexity of MIRNA genes, the importance of introns in the biogenesis and regulation of miRNAs, and raise questions about the processes and molecular mechanisms that drive MIRNA evolution.
PMCID: PMC3581712  PMID: 23341152
alternative splicing; microRNA; root; abscisic acid; plant development; pri-miRNA
3.  Subnuclear compartmentalization of transiently expressed polyadenylated pri-microRNAs 
Cell cycle (Georgetown, Tex.)  2009;8(3):345-356.
MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate expression of their target messenger RNAs. We recently demonstrated that primary miRNA transcripts (pri-miRNAs) retained at transcription sites are processed with enhanced efficiency, suggesting that pri-miRNA processing is coupled to transcription in mammalian cells. We also observed that transiently expressed pri-miRNAs accumulate in nuclear foci with splicing factor SC35 and Microprocessor components, Drosha and DGCR8. Here, we show that pri-miRNAs containing a self-cleaving hepatitis delta ribozyme accumulate in the nucleoplasm after release from their transcription sites, but are not efficiently processed. Pri-miRNAs with ribozyme-generated 3′ ends do not localize to SC35-containing foci, whereas cleaved and polyadenylated pri-miRNA transcripts with or without the pre-miRNA hairpin do. Pri-miRNA/SC35 foci contain a number of proteins normally associated with SC35 domains, including ASF/SF2, PABII, and the prolyl isomerase, Pin1. In contrast, RNA polymerase II and PM/Scl-100 do not strongly colocalize with pri-miRNAs in SC35-containing foci. These data argue that pri-miRNA/SC35-containing foci are not major sites of pri-miRNA processing and that pri-miRNA processing is coupled to transcription. We discuss the implications of our findings relative to recent insights into miRNA biogenesis, mRNA metabolism, and the nuclear organization of gene expression.
PMCID: PMC3004524  PMID: 19177009
microRNA; pri-miRNA; SC35; Drosha; cotranscriptional
4.  Functional links between clustered microRNAs: suppression of cell-cycle inhibitors by microRNA clusters in gastric cancer 
Nucleic Acids Research  2009;37(5):1672-1681.
microRNAs (miRNAs) play integral roles in diverse processes including tumorigenesis. miRNA gene loci are often found in close conjunction, and such clustered miRNA genes are transcribed from a common promoter to generate polycistronic primary transcript. The primary transcript (pri-miRNA) is then processed by two RNase III proteins to release the mature miRNAs. Although it has been speculated that the miRNAs in the same cluster may play related biological functions, this has not been experimentally addressed. Here we report that the miRNAs in two clusters (miR-106b∼93 ∼ 25 and miR-222 ∼ 221) suppress the Cip/Kip family members of Cdk inhibitors (p57Kip2, p21Cip1 and p27Kip1). We show that miR-25 targets p57 through the 3′-UTR. Furthermore, miR-106b and miR-93 control p21 while miR-222 and miR-221 regulate both p27 and p57. Ectopic expression of these miRNAs results in activation of Cdk2 and facilitation of G1/S phase transition. Consistent with these results, both clusters are abnormally upregulated in gastric cancer tissues compared to the corresponding normal tissues. Ectopic expression of miR-222 cluster enhanced tumor growth in the mouse xenograft model. Our study demonstrates the functional associations between clustered miRNAs and further implicates that effective cancer treatment may require a combinatorial approach to target multiple oncogenic miRNA clusters.
PMCID: PMC2655672  PMID: 19153141
5.  Targeted Inhibition of miRNA Maturation with Morpholinos Reveals a Role for miR-375 in Pancreatic Islet Development 
PLoS Biology  2007;5(8):e203.
Several vertebrate microRNAs (miRNAs) have been implicated in cellular processes such as muscle differentiation, synapse function, and insulin secretion. In addition, analysis of Dicer null mutants has shown that miRNAs play a role in tissue morphogenesis. Nonetheless, only a few loss-of-function phenotypes for individual miRNAs have been described to date. Here, we introduce a quick and versatile method to interfere with miRNA function during zebrafish embryonic development. Morpholino oligonucleotides targeting the mature miRNA or the miRNA precursor specifically and temporally knock down miRNAs. Morpholinos can block processing of the primary miRNA (pri-miRNA) or the pre-miRNA, and they can inhibit the activity of the mature miRNA. We used this strategy to knock down 13 miRNAs conserved between zebrafish and mammals. For most miRNAs, this does not result in visible defects, but knockdown of miR-375 causes defects in the morphology of the pancreatic islet. Although the islet is still intact at 24 hours postfertilization, in later stages the islet cells become scattered. This phenotype can be recapitulated by independent control morpholinos targeting other sequences in the miR-375 precursor, excluding off-target effects as cause of the phenotype. The aberrant formation of the endocrine pancreas, caused by miR-375 knockdown, is one of the first loss-of-function phenotypes for an individual miRNA in vertebrate development. The miRNA knockdown strategy presented here will be widely used to unravel miRNA function in zebrafish.
Author Summary
The striking tissue-specific expression patterns of microRNAs (miRNAs) suggest that they play a role in tissue development. These small RNA molecules (∼22 bases in length) are processed from long primary transcripts (pri-miRNA) and regulate gene expression at the posttranscriptional level. There are hundreds of different miRNAs, many of which are strongly conserved. Vertebrate embryonic development is most easily studied in zebrafish, but genetically disrupting miRNA genes to see which miRNA does what is technically challenging. In this study, we interfere with miRNA function during the first few days of zebrafish embryonic development by introducing specific antisense morpholino oligonucleotides (morpholinos have been used previously to interfere with the synthesis of the much larger mRNAs). We show that morpholinos targeting the miRNA precursor can block processing of the pri-miRNA or directly inhibit the activity of the mature miRNA. We also used morpholinos to study the developmental effects of miRNA knockdown. Although we did not observe gross phenotypic defects for many miRNAs, we found that zebrafish miR-375 is essential for formation of the insulin-secreting pancreatic islet. Loss of miR-375 results in dispersed islet cells by 36 hours postfertilization, representing one of the first vertebrate miRNA loss-of-function phenotypes.
The authors show that morpholinos can be used to knock down zebrafish miRNAs, revealing that miR-375 is important for vertebrate pancreas development.
PMCID: PMC1925136  PMID: 17676975
6.  The QKI-5 and QKI-6 RNA Binding Proteins Regulate the Expression of MicroRNA 7 in Glial Cells 
Molecular and Cellular Biology  2013;33(6):1233-1243.
The quaking (qkI) gene encodes 3 major alternatively spliced isoforms that contain unique sequences at their C termini dictating their cellular localization. QKI-5 is predominantly nuclear, whereas QKI-6 is distributed throughout the cell and QKI-7 is cytoplasmic. The QKI isoforms are sequence-specific RNA binding proteins expressed mainly in glial cells modulating RNA splicing, export, and stability. Herein, we identify a new role for the QKI proteins in the regulation of microRNA (miRNA) processing. We observed that small interfering RNA (siRNA)-mediated QKI depletion of U343 glioblastoma cells leads to a robust increase in miR-7 expression. The processing from primary to mature miR-7 was inhibited in the presence QKI-5 and QKI-6 but not QKI-7, suggesting that the nuclear localization plays an important role in the regulation of miR-7 expression. The primary miR-7-1 was bound by the QKI isoforms in a QKI response element (QRE)-specific manner. We observed that the pri-miR-7-1 RNA was tightly bound to Drosha in the presence of the QKI isoforms, and this association was not observed in siRNA-mediated QKI or Drosha-depleted U343 glioblastoma cells. Moreover, the presence of the QKI isoforms led to an increase presence of pri-miR-7 in nuclear foci, suggesting that pri-miR-7-1 is retained in the nucleus by the QKI isoforms. miR-7 is known to target the epidermal growth factor (EGF) receptor (EGFR) 3′ untranslated region (3′-UTR), and indeed, QKI-deficient U343 cells had reduced EGFR expression and decreased ERK activation in response to EGF. Elevated levels of miR-7 are associated with cell cycle arrest, and it was observed that QKI-deficient U343 that harbor elevated levels of miR-7 exhibited defects in cell proliferation that were partially rescued by the addition of a miR-7 inhibitor. These findings suggest that the QKI isoforms regulate glial cell function and proliferation by regulating the processing of certain miRNAs.
PMCID: PMC3592017  PMID: 23319046
7.  A component of the mir-17-92 polycistronic oncomir promotes oncogene-dependent apoptosis 
eLife  2013;2:e00822.
mir-17-92, a potent polycistronic oncomir, encodes six mature miRNAs with complex modes of interactions. In the Eμ-myc Burkitt’s lymphoma model, mir-17-92 exhibits potent oncogenic activity by repressing c-Myc-induced apoptosis, primarily through its miR-19 components. Surprisingly, mir-17-92 also encodes the miR-92 component that negatively regulates its oncogenic cooperation with c-Myc. This miR-92 effect is, at least in part, mediated by its direct repression of Fbw7, which promotes the proteosomal degradation of c-Myc. Thus, overexpressing miR-92 leads to aberrant c-Myc increase, imposing a strong coupling between excessive proliferation and p53-dependent apoptosis. Interestingly, miR-92 antagonizes the oncogenic miR-19 miRNAs; and such functional interaction coordinates proliferation and apoptosis during c-Myc-induced oncogenesis. This miR-19:miR-92 antagonism is disrupted in B-lymphoma cells that favor a greater increase of miR-19 over miR-92. Altogether, we suggest a new paradigm whereby the unique gene structure of a polycistronic oncomir confers an intricate balance between oncogene and tumor suppressor crosstalk.
eLife digest
The role of genes, in very simple terms, is to be transcribed into messenger RNA molecules, which are then translated into strings of amino acids that fold into proteins. Each of these steps is extremely complex, and a wide range of other molecules can speed up, slow down, stop or otherwise disrupt the expression of genes as protein products. Genes can also code for nucleic acids that are not translated into proteins, such as microRNAs. These are small RNA molecules that can reduce the production of proteins by repressing the translation step and/or by partially degrading the messenger RNA molecules.
mir-17-92 is a gene that exemplifies much of this complexity. It codes for six different microRNAs in a single primary transcript, and has been implicated in a number of cancers, including lung cancer, Burkitt’s lymphoma and other forms of lymphomas and leukemia. One of six microRNAs has a longer evolutionary history than the remaining five: mir-92 is found in vertebrates, chordates and invertebrates, whereas the other five are only found in vertebrates. However, it is not known how or why the mir-17-92 gene evolved to code for multiple different microRNAs.
Olive et al. have studied how these mir-17-92 microRNAs functionally interact in mice with Burkitt’s lymphoma, a form of cancer that is associated with a gene called c-Myc being over-activated. Mutations in this gene promote the proliferation of cells, and in cooperation with other genetic lesions, this ultimately leads to cancer. mir-17-92 is implicated in this cancer because it represses the process of programmed cell death (which is induced by the protein c-Myc) that the body employs to stop tumors growing.
Olive et al. found that deleting one of the six microRNAs, miR-92, increased the tendency of the mir-17-92 gene to promote Burkitt’s lymphoma. By repressing an enzyme called Fbw7, miR-92 causes high levels of c-Myc to be produced. While this leads to the uncontrolled proliferation of cells that promotes cancer, it also increases programmed cell death, at least in part, by activating the p53 pathway, a well-known tumor suppression pathway. The experiments also revealed that the action of miR-92 and that of one of the other microRNAs, miR-19, were often opposed to each other. These findings have revealed an unexpected interaction among different components within a single microRNA gene, which acts to maintain an intricate balance between pathways that promote and suppress cancer.
PMCID: PMC3796314  PMID: 24137534
microRNAs; c-Myc; Eμ-myc lymphoma; apoptosis; p53; Mouse
8.  POWERDRESS and Diversified Expression of the MIR172 Gene Family Bolster the Floral Stem Cell Network 
PLoS Genetics  2013;9(1):e1003218.
Termination of the stem cells in the floral meristem (also known as floral determinacy) is critical for the reproductive success of plants, and the molecular activities regulating floral determinacy are precisely orchestrated during the course of floral development. In Arabidopsis thaliana, regulators of floral determinacy include several transcription factor genes, such as APETALA2 (AP2), AGAMOUS (AG), SUPERMAN (SUP), and CRABSCLAW (CRC), as well as a microRNA (miRNA), miR172, which targets AP2. How the transcription factor and miRNA genes are coordinately regulated to achieve floral determinacy is unknown. A mutation in POWERDRESS (PWR), a previously uncharacterized gene encoding a SANT-domain-containing protein, was isolated in this study as an enhancer of the weakly indeterminate ag-10 allele. PWR was found to promote the transcription of CRC, MIR172a, b, and c and/or enhance Pol II occupancy at their promoters, without affecting MIR172d or e. A mutation in mature miR172d was additionally found to enhance the determinacy defects of ag-10 in an AP2-dependent manner, providing direct evidence that miR172d is functional in repressing AP2 and thereby contributes to floral determinacy. Thus, while PWR promotes floral determinacy by enhancing the expression of three of the five MIR172 members as well as CRC, MIR172d, whose expression is PWR-independent, also functions in floral stem cell termination. Taken together, these findings demonstrate how transcriptional diversification and functional redundancy of a miRNA family along with PWR-mediated co-regulation of miRNA and transcription factor genes contribute to the robustness of the floral determinacy network.
Author Summary
microRNAs (miRNAs) are 20–24 nucleotide RNAs that play regulatory roles in many developmental processes in plants and animals. Some miRNAs are encoded by multi-member gene families, and the members may exhibit differential expression patterns. However, the basis of this expression diversification and its developmental impact are poorly understood. By studying miR172, which represses its target APETALA2 (AP2) and thereby promotes the determinate growth of flowers (also known as floral determinacy), we show that the five MIR172 genes undergo differential transcriptional regulation. POWERDRESS (PWR), a previously uncharacterized SANT-domain-containing protein, promotes floral determinacy by enhancing the expression of MIR172a-c. MIR172d, whose expression is PWR-independent, was found to be functional in floral determinacy by repressing AP2. PWR also promotes floral determinacy through a transcription factor previously implicated in this process. Thus, transcriptional diversification of a miRNA family and PWR-mediated co-regulation of miRNA and transcription factor genes involved in floral determinacy contribute to the robustness of this developmental network.
PMCID: PMC3547843  PMID: 23349639
9.  Feed-Forward Microprocessing and Splicing Activities at a MicroRNA–Containing Intron 
PLoS Genetics  2011;7(10):e1002330.
The majority of mammalian microRNA (miRNA) genes reside within introns of protein-encoding and non-coding genes, yet the mechanisms coordinating primary transcript processing into both mature miRNA and spliced mRNA are poorly understood. Analysis of melanoma invasion suppressor miR-211 expressed from intron 6 of melastatin revealed that microprocessing of miR-211 promotes splicing of the exon 6–exon 7 junction of melastatin by a mechanism requiring the RNase III activity of Drosha. Additionally, mutations in the 5′ splice site (5′SS), but not in the 3′SS, branch point, or polypyrimidine tract of intron 6 reduced miR-211 biogenesis and Drosha recruitment to intron 6, indicating that 5′SS recognition by the spliceosome promotes microprocessing of miR-211. Globally, knockdown of U1 splicing factors reduced intronic miRNA expression. Our data demonstrate novel mutually-cooperative microprocessing and splicing activities at an intronic miRNA locus and suggest that the initiation of spliceosome assembly may promote microprocessing of intronic miRNAs.
Author Summary
MicroRNA (miRNA) genes are transcribed as long primary RNAs containing local hairpins that are excised by the Microprocessor complex minimally composed of Drosha and DGCR8. Most mammalian miRNAs reside in introns of protein-encoding and non-coding genes, but it is unclear how microprocessing of an intronic miRNA and splicing at the host gene intron affect each other. We recently reported that in melanoma, a miRNA expressed from intron 6 of melastatin (miR-211) assumes the tumor suppressive function of its host gene. In our current work, we detected elevated melastatin exon 6–exon 7 junctions relative to other exon-exon junctions that lack intronic miRNAs, suggesting that microprocessing promotes splicing. We show that microprocessing of miR-211 precedes completion of splicing of the exon 6–exon 7 junctions and that Drosha's endonuclease activity is required to facilitate exon 6–exon 7 junction formation. Additionally, we found that the first step of spliceosome assembly, recognition of the 5′ splice site by the U1 snRNP complex, promotes microprocessing of miR-211 and other intronic but not intergenic miRNAs. Our findings reveal a mutually cooperative, physical, and functional coupling of intronic miRNA biogenesis and splicing at the host intron, and they suggest a global positive effect of spliceosome assembly on intronic miRNA microprocessing.
PMCID: PMC3197686  PMID: 22028668
10.  Gene Regulation in Giardia lambia Involves a Putative MicroRNA Derived from a Small Nucleolar RNA 
Two core microRNA (miRNA) pathway proteins, Dicer and Argonaute, are found in Giardia lamblia, a deeply branching parasitic protozoan. There are, however, no apparent homologues of Drosha or Exportin5 in the genome. Here, we report a 26 nucleotide (nt) RNA derived from a 106 nt Box C/D snoRNA, GlsR2. This small RNA, designated miR5, localizes to the 3′ end of GlsR2 and has a 75 nt hairpin precursor. GlsR2 is processed by the Dicer from Giardia (GlDcr) and generated miR5. Immunoprecipitation of the Argonaute from Giardia (GlAgo) brought down miR5. When a Renilla Luciferase transcript with a 26 nt miR5 antisense sequence at the 3′-untranslated region (3′ UTR) was introduced into Giardia trophozoites, Luciferase expression was reduced ∼25% when synthetic miR5 was also introduced. The Luciferase mRNA level remained, however, unchanged, suggesting translation repression by miR5. This inhibition was fully reversed by introducing also a 2′-O-methylated antisense inhibitor of miR5, suggesting that miR5 acts by interacting specifically with the antisense sequence in the mRNA. A partial antisense knock down of GlDcr or GlAgo in Giardia indicated that the former is needed for miR5 biogenesis whereas the latter is required for miR5-mediated translational repression. Potential targets for miR5 with canonical seed sequences were predicted bioinformatically near the stop codon of Giardia mRNAs. Four out of the 21 most likely targets were tested in the Luciferase reporter assay. miR5 was found to inhibit Luciferase expression (∼20%) of transcripts carrying these potential target sites, indicating that snoRNA-derived miRNA can regulate the expression of multiple genes in Giardia.
Author Summary
Giardia lambia is a deeply branched parasitic protozoan and the pathogen causing the diarrhetic disorder, giardiasis. The mechanism of gene regulation in this organism is largely unknown. Here, we identified a 26 nucleotide (nt) small RNA from the 3′-end of a 106 nt small nucleolar RNA (GlsR2) in Giardia. GlsR2 is processed through the action of a Dicer protein in Giardia to generate the 26 nt RNA. The latter becomes associated with the Argonaute protein. The protein-RNA complex can repress the translation of messenger RNAs carrying the antisense sequence of the 26 nt RNA at the 3′-untranslated region. This small RNA, designated microRNA5 (miR5), has several potential targets identified in Giardia, among which four were further tested in Giardia and found their translation repressed by miR5. This is the second functioning microRNA we have indentified in Giardia. The microRNAs could be thus important regulators of gene expression in this ancient single cellular organism.
PMCID: PMC3196473  PMID: 22028939
11.  microRNA-122 as a regulator of mitochondrial metabolic gene network in hepatocellular carcinoma 
A moderate loss of miR-122 function correlates with up-regulation of seed-matched genes and down-regulation of mitochondrially localized genes in both human hepatocellular carcinoma and in normal mice treated with anti-miR-122 antagomir.Putative direct targets up-regulated with loss of miR-122 and secondary targets down-regulated with loss of miR-122 are conserved between human beings and mice and are rapidly regulated in vitro in response to miR-122 over- and under-expression.Loss of miR-122 secondary target expression in either tumorous or adjacent non-tumorous tissue predicts poor survival of heptatocellular carcinoma patients.
Hepatocellular carcinoma (HCC) is one of the most aggressive human malignancies, common in Asia, Africa, and in areas with endemic infections of hepatitis-B or -C viruses (HBV or HCV) (But et al, 2008). Globally, the 5-year survival rate of HCC is <5% and about 600 000 HCC patients die each year. The high mortality associated with this disease is mainly attributed to the failure to diagnose HCC patients at an early stage and a lack of effective therapies for patients with advanced stage HCC. Understanding the relationships between phenotypic and molecular changes in HCC is, therefore, of paramount importance for the development of improved HCC diagnosis and treatment methods.
In this study, we examined mRNA and microRNA (miRNA)-expression profiles of tumor and adjacent non-tumor liver tissue from HCC patients. The patient population was selected from a region of endemic HBV infection, and HBV infection appears to contribute to the etiology of HCC in these patients. A total of 96 HCC patients were included in the study, of which about 88% tested positive for HBV antigen; patients testing positive for HCV antigen were excluded. Among the 220 miRNAs profiled, miR-122 was the most highly expressed miRNA in liver, and its expression was decreased almost two-fold in HCC tissue relative to adjacent non-tumor tissue, confirming earlier observations (Lagos-Quintana et al, 2002; Kutay et al, 2006; Budhu et al, 2008).
Over 1000 transcripts were correlated and over 1000 transcripts were anti-correlated with miR-122 expression. Consistent with the idea that transcripts anti-correlated with miR-122 are potential miR-122 targets, the most highly anti-correlated transcripts were highly enriched for the presence of the miR-122 central seed hexamer, CACTCC, in the 3′UTR. Although the complete set of negatively correlated genes was enriched for cell-cycle genes, the subset of seed-matched genes had no significant KEGG Pathway annotation, suggesting that miR-122 is unlikely to directly regulate the cell cycle in these patients. In contrast, transcripts positively correlated with miR-122 were not enriched for 3′UTR seed matches to miR-122. Interestingly, these 1042 transcripts were enriched for genes coding for mitochondrially localized proteins and for metabolic functions.
To analyze the impact of loss of miR-122 in vivo, silencing of miR-122 was performed by antisense inhibition (anti-miR-122) in wild-type mice (Figure 3). As with the genes negatively correlated with miR-122 in HCC patients, no significant biological annotation was associated with the seed-matched genes up-regulated by anti-miR-122 in mouse livers. The most significantly enriched biological annotation for anti-miR-122 down-regulated genes, as for positively correlated genes in HCC, was mitochondrial localization; the down-regulated mitochondrial genes were enriched for metabolic functions. Putative direct and downstream targets with orthologs on both the human and mouse microarrays showed significant overlap for regulations in the same direction. These overlaps defined sets of putative miR-122 primary and secondary targets. The results were further extended in the analysis of a separate dataset from 180 HCC, 40 cirrhotic, and 6 normal liver tissue samples (Figure 4), showing anti-correlation of proposed primary and secondary targets in non-healthy tissues.
To validate the direct correlation between miR-122 and some of the primary and secondary targets, we determined the expression of putative targets after transfection of miR-122 mimetic into PLC/PRF/5 HCC cells, including the putative direct targets SMARCD1 and MAP3K3 (MEKK3), a target described in the literature, CAT-1 (SLC7A1), and three putative secondary targets, PPARGC1A (PGC-1α) and succinate dehydrogenase subunits A and B. As expected, the putative direct targets showed reduced expression, whereas the putative secondary target genes showed increased expression in cells over-expressing miR-122 (Figure 4).
Functional classification of genes using the total ancestry method (Yu et al, 2007) identified PPARGC1A (PGC-1α) as the most connected secondary target. PPARGC1A has been proposed to function as a master regulator of mitochondrial biogenesis (Ventura-Clapier et al, 2008), suggesting that loss of PPARGC1A expression may contribute to the loss of mitochondrial gene expression correlated with loss of miR-122 expression. To further validate the link of miR-122 and PGC-1α protein, we transfected PLC/PRF/5 cells with miR-122-expression vector, and observed an increase in PGC-1α protein levels. Importantly, transfection of both miR-122 mimetic and miR-122-expression vector significantly reduced the lactate content of PLC/PRF/5 cells, whereas anti-miR-122 treatment increased lactate production. Together, the data support the function of miR-122 in mitochondrial metabolic functions.
Patient survival was not directly associated with miR-122-expression levels. However, miR-122 secondary targets were expressed at significantly higher levels in both tumor and adjacent non-tumor tissues among survivors as compared with deceased patients, providing supporting evidence for the potential relevance of loss of miR-122 function in HCC patient morbidity and mortality.
Overall, our findings reveal potentially new biological functions for miR-122 in liver physiology. We observed decreased expression of miR-122, a liver-specific miRNA, in HBV-associated HCC, and loss of miR-122 seemed to correlate with the decrease of mitochondrion-related metabolic pathway gene expression in HCC and in non-tumor liver tissues, a result that is consistent with the outcome of treatment of mice with anti-miR-122 and is of prognostic significance for HCC patients. Further investigation will be conducted to dissect the regulatory function of miR-122 on mitochondrial metabolism in HCC and to test whether increasing miR-122 expression can improve mitochondrial function in liver and perhaps in liver tumor tissues. Moreover, these results support the idea that primary targets of a given miRNA may be distributed over a variety of functional categories while resulting in a coordinated secondary response, potentially through synergistic action (Linsley et al, 2007).
Tumorigenesis involves multistep genetic alterations. To elucidate the microRNA (miRNA)–gene interaction network in carcinogenesis, we examined their genome-wide expression profiles in 96 pairs of tumor/non-tumor tissues from hepatocellular carcinoma (HCC). Comprehensive analysis of the coordinate expression of miRNAs and mRNAs reveals that miR-122 is under-expressed in HCC and that increased expression of miR-122 seed-matched genes leads to a loss of mitochondrial metabolic function. Furthermore, the miR-122 secondary targets, which decrease in expression, are good prognostic markers for HCC. Transcriptome profiling data from additional 180 HCC and 40 liver cirrhotic patients in the same cohort were used to confirm the anti-correlation of miR-122 primary and secondary target gene sets. The HCC findings can be recapitulated in mouse liver by silencing miR-122 with antagomir treatment followed by gene-expression microarray analysis. In vitro miR-122 data further provided a direct link between induction of miR-122-controlled genes and impairment of mitochondrial metabolism. In conclusion, miR-122 regulates mitochondrial metabolism and its loss may be detrimental to sustaining critical liver function and contribute to morbidity and mortality of liver cancer patients.
PMCID: PMC2950084  PMID: 20739924
hepatocellular carcinoma; microarray; miR-122; mitochondrial; survival
12.  Functional Specialization of the Plant miR396 Regulatory Network through Distinct MicroRNA–Target Interactions 
PLoS Genetics  2012;8(1):e1002419.
MicroRNAs (miRNAs) are ∼21 nt small RNAs that regulate gene expression in animals and plants. They can be grouped into families comprising different genes encoding similar or identical mature miRNAs. Several miRNA families are deeply conserved in plant lineages and regulate key aspects of plant development, hormone signaling, and stress response. The ancient miRNA miR396 regulates conserved targets belonging to the GROWTH-REGULATING FACTOR (GRF) family of transcription factors, which are known to control cell proliferation in Arabidopsis leaves. In this work, we characterized the regulation of an additional target for miR396, the transcription factor bHLH74, that is necessary for Arabidopsis normal development. bHLH74 homologs with a miR396 target site could only be detected in the sister families Brassicaceae and Cleomaceae. Still, bHLH74 repression by miR396 is required for margin and vein pattern formation of Arabidopsis leaves. MiR396 contributes to the spatio-temporal regulation of GRF and bHLH74 expression during leaf development. Furthermore, a survey of miR396 sequences in different species showed variations in the 5′ portion of the miRNA, a region known to be important for miRNA activity. Analysis of different miR396 variants in Arabidopsis thaliana revealed that they have an enhanced activity toward GRF transcription factors. The interaction between the GRF target site and miR396 has a bulge between positions 7 and 8 of the miRNA. Our data indicate that such bulge modulates the strength of the miR396-mediated repression and that this modulation is essential to shape the precise spatio-temporal pattern of GRF2 expression. The results show that ancient miRNAs can regulate conserved targets with varied efficiency in different species, and we further propose that they could acquire new targets whose control might also be biologically relevant.
Author Summary
Plants and other multicellular organisms need precise spatio-temporal control of gene expression, and this regulatory capacity depends, in part, on small RNAs. MicroRNAs (miRNAs) are one class of ∼21 nt small RNAs that originate from endogenous fold-back precursors found in plants and animals. They recognize complementary target sites in target mRNAs and guide them to cleavage or translational arrest. Studies of conserved miRNA networks in Arabidopsis and other plants have revealed that they fulfill essential regulatory roles. Most of the ancient miRNAs regulate transcription factors involved in plant development and hormone signaling. Here, we characterize the miR396 regulatory network. While miR396 regulates GRF transcription factors, at least in angiosperms and gymnosperms, this miRNA additionally regulates another transcription factor of the bHLH class but only in Arabidopsis thaliana and closely related species. Most conspicuously, the regulation of both conserved and new targets is important for leaf development in Arabidopsis. We also show that miRNA variants can exist in certain species and that they can display an enhanced activity towards their targets. In summary, we propose that conserved miRNA regulatory networks might expand their functions by the recruitment of additional targets as well as by slight variations in the small RNA sequences.
PMCID: PMC3252272  PMID: 22242012
13.  Identification of Nuclear and Cytoplasmic mRNA Targets for the Shuttling Protein SF2/ASF 
PLoS ONE  2008;3(10):e3369.
The serine and arginine-rich protein family (SR proteins) are highly conserved regulators of pre-mRNA splicing. SF2/ASF, a prototype member of the SR protein family, is a multifunctional RNA binding protein with roles in pre-mRNA splicing, mRNA export and mRNA translation. These observations suggest the intriguing hypothesis that SF2/ASF may couple splicing and translation of specific mRNA targets in vivo. Unfortunately the paucity of endogenous mRNA targets for SF2/ASF has hindered testing of this hypothesis. Here, we identify endogenous mRNAs directly cross-linked to SF2/ASF in different sub-cellular compartments. Cross-Linking Immunoprecipitation (CLIP) captures the in situ specificity of protein-RNA interaction and allows for the simultaneous identification of endogenous RNA targets as well as the locations of binding sites within the RNA transcript. Using the CLIP method we identified 326 binding sites for SF2/ASF in RNA transcripts from 180 protein coding genes. A purine-rich consensus motif was identified in binding sites located within exon sequences but not introns. Furthermore, 72 binding sites were occupied by SF2/ASF in different sub-cellular fractions suggesting that these binding sites may influence the splicing or translational control of endogenous mRNA targets. We demonstrate that ectopic expression of SF2/ASF regulates the splicing and polysome association of transcripts derived from the SFRS1, PABC1, NETO2 and ENSA genes. Taken together the data presented here indicate that SF2/ASF has the capacity to co-regulate the nuclear and cytoplasmic processing of specific mRNAs and provide further evidence that the nuclear history of an mRNA may influence its cytoplasmic fate.
PMCID: PMC2556390  PMID: 18841201
14.  Genetic variability of microRNA regulome in human 
MicroRNAs are currently being extensively studied due to their important role as post-transcriptional regulators. During miRNA biogenesis, precursors undergo two cleavage steps performed by Drosha-DGCR8 (Microprocessor) cleaving of pri-miRNA to produce pre-miRNA and Dicer-mediated cleaving to create mature miRNA. Genetic variants within human miRNA regulome have been shown to influence miRNA expression, target interaction and to affect the phenotype. In this study, we reviewed the literature, existing bioinformatics tools and catalogs associated with polymorphic miRNA regulome, and organized them into four categories: (1) polymorphisms located within miRNA genes (miR-SNPs), (2) transcription factor-binding sites/miRNA regulatory regions (miR-rSNPs), (3) miRNA target sites (miR-TS-SNPs), and 4. miRNA silencing machinery (miR-SM-SNPs). Since the miR-SM-SNPs have not been systematically studied yet, we have collected polymorphisms associated with miRNA silencing machinery. We have developed two catalogs containing genetic variability within: (1) genes encoding three main catalytic components of the silencing machinery, DROSHA, DGCR8, and DICER1; (2) miRNA genes itself, overlapping Drosha and Dicer cleavage sites. The developed resource of polymorphisms is available online ( and will be useful for further functional studies and development of biomarkers associated with diseases and phenotypic traits.
PMCID: PMC4299713  PMID: 25629077
Biogenesis; Dicer; Drosha; human; microRNA (miRNA); polymorphisms
15.  An in silico analysis of dynamic changes in microRNA expression profiles in stepwise development of nasopharyngeal carcinoma 
MicroRNAs (miRNAs) are small non-coding RNAs that participate in the spatiotemporal regulation of messenger RNA (mRNA) and protein synthesis. Recent studies have shown that some miRNAs are involved in the progression of nasopharyngeal carcinoma (NPC). However, the aberrant miRNAs implicated in different clinical stages of NPC remain unknown and their functions have not been systematically studied.
In this study, miRNA microarray assay was performed on biopsies from different clinical stages of NPC. TargetScan was used to predict the target genes of the miRNAs. The target gene list was narrowed down by searching the data from the UniGene database to identify the nasopharyngeal-specific genes. The data reduction strategy was used to overlay with nasopharyngeal-specifically expressed miRNA target genes and complementary DNA (cDNA) expression data. The selected target genes were analyzed in the Gene Ontology (GO) biological process and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway. The microRNA-Gene-Network was build based on the interactions of miRNAs and target genes. miRNA promoters were analyzed for the transcription factor (TF) binding sites. UCSC Genome database was used to construct the TF-miRNAs interaction networks.
Forty-eight miRNAs with significant change were obtained by Multi-Class Dif. The most enriched GO terms in the predicted target genes of miRNA were cell proliferation, cell migration and cell matrix adhesion. KEGG analysis showed that target genes were significantly involved in adherens junction, cell adhesion molecules, p53 signalling pathway et al. Comprehensive analysis of the coordinate expression of miRNAs and mRNAs reveals that miR-29a/c, miR-34b, miR-34c-3p, miR-34c-5p, miR-429, miR-203, miR-222, miR-1/206, miR-141, miR-18a/b, miR-544, miR-205 and miR-149 may play important roles on the development of NPC. We proposed an integrative strategy for identifying the miRNA-mRNA regulatory modules and TF-miRNA regulatory networks. TF including ETS2, MYB, Sp1, KLF6, NFE2, PCBP1 and TMEM54 exert regulatory functions on the miRNA expression.
This study provides perspective on the microRNA expression during the development of NPC. It revealed the global trends in miRNA interactome in NPC. It concluded that miRNAs might play important regulatory roles through the target genes and transcription factors in the stepwise development of NPC.
PMCID: PMC3293045  PMID: 22260379
16.  A microRNA network functioning in the regulation of radiobiological effects 
Journal of Radiation Research  2014;55(Suppl 1):i57-i58.
MicroRNA (miRNA), a small non-coding RNA molecule, is vital in genetic regulation, and miRNA pathway, which regulates gene expression through degradation or translational suppression of their target transcripts, is highly conservative in evolution.
Although profiles of miRNAs are different in various cell types and tissues, miRNAs have been considered as a crucial class of regulators in cellular response to ionizing radiation (IR). By carrying out a series of experiments, we have found that altered transcriptional regulation network composed of radiation-mediated miRNAs regulates the expression of their downstream target genes in most biological processes to control cell growth, cell cycle and apoptosis. For example, the newly identified miR-3928 negatively regulates the expression of Dicer, which has been validated by the luciferase assay and western blotting. Dicer is not only a key participant in responding to radiation, but also a critical factor for the maturation of miRNAs, suggesting that miR-3928 affects on the expression of other miRNAs through regulating Dicer. Among the miRNAs controlled by the Dicer, we reveal that miR-185 and miR-663 can efficiently suppress ATR and TGF-β1 expression, which are both important responders in the process of radiobiological effects. Further experiments reveal that the expression of Dicer is suppressed by miR-3928 induced by IR and consequently, the maturation of other miRNAs including miR-185 and miR-663 is inhibited, resulting in the abundantly enhanced expression of ATR and TGF-β1 respectively. This mechanism to hammer at fixing DNA damage or promote cells to apoptosis caused by IR has important implications in the decision of cell fates.
Moreover, it has been shown that the expression of BTG1 is characterized in response to factors that induce growth arrest and subsequent differentiation both in vivo and in vitro, affecting cellular physiological progresses of angiogenesis, follicular development and myoblast and B cell differentiation, through regulating cell growth, migration, cell cycle, apoptosis and differentiation. BTG1 gene is phylogenetically highly conserved in its coding and 3′-untranslated region (UTR), which is considered as a decisive element involved in regulation of BTG1 expression. We present evidence that BTG1 can be induced by IR and confirm that miR-454-3p, whose gene locates in the intron of Ska2 gene, can regulate BTG1 expression through directly binding to the 3′-UTR of BTG1 mRNA. These results point out that increased expression of BTG1 caused by the down-regulation of miR-454-3p in case that IR modulates endogenous activity of PRMT1, a BTG1-binding partner, which can methylate endogenous transcription factors to change gene expression pattern and reply radiostilumation. An inverse relationship between the levels of expression of BTG1 and miR-454-3p reveals that there exists a new pathway in response to IR stimulation. Furthermore, cell growth will be transiently increased by the knockdown of BTG1 by transfecting BTG1 siRNA or miR-454-3p mimic. However, the apoptotic state of cells can be tested after 2 days. Down-regulation of BTG1 by miR-454-3p increases the sensitivity of human renal cell carcinoma 786-O cells to IR-induced apoptosis, suggesting that BTG1 could serve as a terget for sensitizing renal carcinoma to standard radiotherapy.
Taken together, all these data indicate that alteration of miRNA expression is evident in the cellular response to IR. MiR-3928, miR-185, miR-663 and miR-454-3p may constitute a complex network contributing to the regulation of radiobiological effects. It is apparent that the study of radiation-related miRNAs is beneficial to qualitatively and quantitatively modulating radiobiological effects, and also in favor of the advanced research of miRNA functions.
PMCID: PMC3941529
microRNA; network; Dicer; BTG1; ionizing radiation
17.  Degradation of Cellular miR-27 by a Novel, Highly Abundant Viral Transcript Is Important for Efficient Virus Replication In Vivo 
PLoS Pathogens  2012;8(2):e1002510.
Cytomegaloviruses express large amounts of viral miRNAs during lytic infection, yet, they only modestly alter the cellular miRNA profile. The most prominent alteration upon lytic murine cytomegalovirus (MCMV) infection is the rapid degradation of the cellular miR-27a and miR-27b. Here, we report that this regulation is mediated by the ∼1.7 kb spliced and highly abundant MCMV m169 transcript. Specificity to miR-27a/b is mediated by a single, apparently optimized, miRNA binding site located in its 3′-UTR. This site is easily and efficiently retargeted to other cellular and viral miRNAs by target site replacement. Expression of the 3′-UTR of m169 by an adenoviral vector was sufficient to mediate its function, indicating that no other viral factors are essential in this process. Degradation of miR-27a/b was found to be accompanied by 3′-tailing and -trimming. Despite its dramatic effect on miRNA stability, we found this interaction to be mutual, indicating potential regulation of m169 by miR-27a/b. Most interestingly, three mutant viruses no longer able to target miR-27a/b, either due to miRNA target site disruption or target site replacement, showed significant attenuation in multiple organs as early as 4 days post infection, indicating that degradation of miR-27a/b is important for efficient MCMV replication in vivo.
Author Summary
MicroRNAs are small, non-coding RNAs which shape and fine-tune gene expression of at least a third of our genes. During millions of years of coevolution with their hosts, herpesviruses have both usurped the host cell miRNA machinery by expressing their own sets of miRNAs, and learned to modify host miRNA expression for their own needs. Recently, we reported on the rapid degradation of two cellular miRNAs upon lytic murine cytomegalovirus (MCMV) infection, namely miR-27a and miR-27b. In this paper, we show that their regulation is mediated by the highly abundant viral transcript m169. It targets miR-27a/b via a single binding site in its 3′-UTR, which can be efficiently retargeted to other cellular and viral miRNAs, enabling the efficient knock-down of individual miRNAs of interest. Degradation of miR-27a/b is preceded by its 3′-tailing and -trimming. Most interestingly, three mutant viruses unable to target miR-27a/b showed significantly lower virus titers in various organs during acute MCMV infection, indicating that degradation of miR-27a/b is important for efficient virus replication in vivo.
PMCID: PMC3276556  PMID: 22346748
18.  Concordant Regulation of Translation and mRNA Abundance for Hundreds of Targets of a Human microRNA 
PLoS Biology  2009;7(11):e1000238.
A specific microRNA reduces the synthesis of hundreds of proteins via concordant effects on the abundance and translation of the mRNAs that encode them.
MicroRNAs (miRNAs) regulate gene expression posttranscriptionally by interfering with a target mRNA's translation, stability, or both. We sought to dissect the respective contributions of translational inhibition and mRNA decay to microRNA regulation. We identified direct targets of a specific miRNA, miR-124, by virtue of their association with Argonaute proteins, core components of miRNA effector complexes, in response to miR-124 transfection in human tissue culture cells. In parallel, we assessed mRNA levels and obtained translation profiles using a novel global approach to analyze polysomes separated on sucrose gradients. Analysis of translation profiles for ∼8,000 genes in these proliferative human cells revealed that basic features of translation are similar to those previously observed in rapidly growing Saccharomyces cerevisiae. For ∼600 mRNAs specifically recruited to Argonaute proteins by miR-124, we found reductions in both the mRNA abundance and inferred translation rate spanning a large dynamic range. The changes in mRNA levels of these miR-124 targets were larger than the changes in translation, with average decreases of 35% and 12%, respectively. Further, there was no identifiable subgroup of mRNA targets for which the translational response was dominant. Both ribosome occupancy (the fraction of a given gene's transcripts associated with ribosomes) and ribosome density (the average number of ribosomes bound per unit length of coding sequence) were selectively reduced for hundreds of miR-124 targets by the presence of miR-124. Changes in protein abundance inferred from the observed changes in mRNA abundance and translation profiles closely matched changes directly determined by Western analysis for 11 of 12 proteins, suggesting that our assays captured most of miR-124–mediated regulation. These results suggest that miRNAs inhibit translation initiation or stimulate ribosome drop-off preferentially near the start site and are not consistent with inhibition of polypeptide elongation, or nascent polypeptide degradation contributing significantly to miRNA-mediated regulation in proliferating HEK293T cells. The observation of concordant changes in mRNA abundance and translational rate for hundreds of miR-124 targets is consistent with a functional link between these two regulatory outcomes of miRNA targeting, and the well-documented interrelationship between translation and mRNA decay.
Author Summary
The human genome contains directions to regulate the timing and magnitude of expression of its thousands of genes. MicroRNAs are important regulatory RNAs that tune the expression levels of tens to hundreds of specific genes by pairing to complimentary stretches in the messenger RNAs from these genes, thereby reducing their stability and their translation into protein. Although the importance of microRNAs is appreciated, little is known about the relative contributions of degradation or repression of translation of the cognate mRNAs to the overall effects on protein synthesis, or the links between these two regulatory mechanisms. We devised a simple, economical method to systematically measure mRNA translation profiles, then applied this method, in combination with gene expression analysis, to measure the effects of the human microRNA miR-124 on the abundance and apparent translation rate of its mRNA targets. We found that for the ∼600 mRNA targets of miR-124 that were identified by their association with microRNA effector complexes, around three quarters of the reduction in estimated protein synthesis was explained by changes in mRNA abundance. Although the apparent changes in translation efficiencies of the targeted mRNAs were smaller in magnitude, they were highly correlated with changes in the abundance of those RNAs, suggesting a functional link between microRNA-mediated repression of translation and mRNA decay.
PMCID: PMC2766070  PMID: 19901979
19.  snoRNA, a Novel Precursor of microRNA in Giardia lamblia 
PLoS Pathogens  2008;4(11):e1000224.
An Argonaute homolog and a functional Dicer have been identified in the ancient eukaryote Giardia lamblia, which apparently lacks the ability to perform RNA interference (RNAi). The Giardia Argonaute plays an essential role in growth and is capable of binding specifically to the m7G-cap, suggesting a potential involvement in microRNA (miRNA)-mediated translational repression. To test such a possibility, small RNAs were isolated from Giardia trophozoites, cloned, and sequenced. A 26-nucleotide (nt) small RNA (miR2) was identified as a product of Dicer-processed snoRNA GlsR17 and localized to the cytoplasm by fluorescence in situ hybridization, whereas GlsR17 was found primarily in the nucleolus of only one of the two nuclei in Giardia. Three other small RNAs were also identified as products of snoRNAs, suggesting that the latter could be novel precursors of miRNAs in Giardia. Putative miR2 target sites were identified at the 3′-untranslated regions (UTR) of 22 variant surface protein mRNAs using the miRanda program. In vivo expression of Renilla luciferase mRNA containing six identical miR2 target sites in the 3′-UTR was reduced by 40% when co-transfected with synthetic miR2, while the level of luciferase mRNA remained unaffected. Thus, miR2 likely affects translation but not mRNA stability. This repression, however, was not observed when Argonaute was knocked down in Giardia using a ribozyme-antisense RNA. Instead, an enhancement of luciferase expression was observed, suggesting a loss of endogenous miR2-mediated repression when this protein is depleted. Additionally, the level of miR2 was significantly reduced when Dicer was knocked down. In all, the evidence indicates the presence of a snoRNA-derived miRNA-mediated translational repression in Giardia.
Author Summary
Gene regulation in Giardia lamblia, a primitive parasitic protozoan responsible for the diarrheal disease giardiasis, is poorly understood. There is no consensus promoter sequence. A simple eight–base pair AT-rich region is sufficient to initiate gene transcription in this organism. Thus, the main control of gene expression may occur after the stage of transcription. The presence of Dicer and Argonaute homologs in Giardia suggested that microRNA (miRNA)-mediated translational repression could be one mechanism of gene regulation. In this work, we characterized the presence of the miRNA pathway in Giardia as well as identified the novel use of small nucleolar RNA (snoRNA) as miRNA precursors. Potential target sites for one small RNA (miR2) were identified with the miRanda program. In vivo reporter assays confirmed the specific interaction between the target sites and miR2. A ribozyme-mediated reduction of Dicer and Argonaute in Giardia showed that the former is required for miR2 production whereas the latter functions in mediating the inhibition of reporter expression, which agrees with the roles of these two proteins. This is the first evidence of miRNA-mediated gene regulation in Giardia and the first demonstration of the use of snoRNAs as miRNA precursors.
PMCID: PMC2583053  PMID: 19043559
20.  Transcriptional mechanism for the paired miR-433 and miR-127 genes by nuclear receptors SHP and ERRγ 
Nucleic Acids Research  2008;36(18):5727-5735.
MicroRNAs (miRNAs, miRs) are genomically encoded small ∼22 nt RNA molecules that have been shown to mediate translational repression of target mRNAs involved in cellular proliferation, differentiation and death. Despite intensive studies on their physiological and pathological functions, the molecular mechanism of how miRNA gene transcription is regulated remains largely unknown. Microarray profiling revealed 21 miRNAs clustered on chromosome 12, including miR-433 and miR-127, that were co-upregulated in small heterodimer partner (SHP, NR0B2) SHP knockouts (SHP–/–) liver. Gene cloning revealed that the 3′-coding region of pri-miR-433 served as the promoter region of pri-miR-127. Estrogen related receptor (ERRγ, NR3B3) robustly activated miR-433 and miR-127 promoter reporters through ERRE, which was transrepressed by SHP. The strong elevation of miR-433 and miR-127 in Hepa-1 cells correlated with the down-regulation of SHP and up-regulation of ERRγ. Ectopic expression of ERRγ induced miR-433 and miR-127 expression, which was repressed by SHP coexpression. In contrast, knockdown ERRγ decreased miR-433 and miR-127 expression. In addition, the ERRγ agonist GSK4716 induced miR-433 and miR-127 expression both in vitro and in vivo, respectively. In summary, the coupled miR-433 and miR-127 genes were transcribed from independent promoters regulated by nuclear receptors ERRγ/SHP in a compact space by using overlapping genomic regions.
PMCID: PMC2566885  PMID: 18776219
21.  Roles for SR Proteins and hnRNP A1 in the Regulation of c-src Exon N1 
Molecular and Cellular Biology  2003;23(6):1874-1884.
The splicing of the c-src exon N1 is controlled by an intricate combination of positive and negative RNA elements. Most previous work on these sequences focused on intronic elements found upstream and downstream of exon N1. However, it was demonstrated that the 5′ half of the N1 exon itself acts as a splicing enhancer in vivo. Here we examine the function of this regulatory element in vitro. We show that a mutation in this sequence decreases splicing of the N1 exon in vitro. Proteins binding to this element were identified as hnRNP A1, hnRNP H, hnRNP F, and SF2/ASF by site-specific cross-linking and immunoprecipitation. The binding of these proteins to the RNA was eliminated by a mutation in the exonic element. The activities of hnRNP A1 and SF2/ASF on N1 splicing were examined by adding purified protein to in vitro splicing reactions. SF2/ASF and another SR protein, SC35, are both able to stimulate splicing of c-src pre-mRNA. However, splicing activation by SF2/ASF is dependent on the N1 exon enhancer element whereas activation by SC35 is not. In contrast to SF2/ASF and in agreement with other systems, hnRNP A1 repressed c-src splicing in vitro. The negative activity of hnRNP A1 on splicing was compared with that of PTB, a protein previously demonstrated to repress splicing in this system. Both proteins repress exon N1 splicing, and both counteract the enhancing activity of the SR proteins. Removal of the PTB binding sites upstream of N1 prevents PTB-mediated repression but does not affect A1-mediated repression. Thus, hnRNP A1 and PTB use different mechanisms to repress c-src splicing. Our results link the activity of these well-known exonic splicing regulators, SF2/ASF and hnRNP A1, to the splicing of an exon primarily controlled by intronic factors.
PMCID: PMC149473  PMID: 12612063
22.  Observation of miRNA Gene Expression in Zebrafish Embryos by In Situ Hybridization to MicroRNA Primary Transcripts 
Zebrafish  2011;8(1):1-8.
MicroRNAs (miRNAs) add a previously unexpected layer to the post-transcriptional regulation of protein production. Although locked nucleic acids (LNAs) reveal the distribution of mature miRNAs by in situ hybridization (ISH) experiments in zebrafish and other organisms, high cost has restricted their use. Further, LNA probes designed to recognize mature miRNAs do not distinguish expression patterns of two miRNA genes that produce the same mature miRNA sequence. Riboprobes are substantially less expensive than LNAs, but have not been used to detect miRNA gene expression because they do not bind with high affinity to the short, 22-nucleotide-long mature miRNAs. To solve these problems, we capitalized on the fact that miRNAs are initially transcribed into long primary transcripts (pri-mRNAs). We show here that conventional digoxigenin-labeled riboprobes can bind to primary miRNA transcripts in zebrafish embryos. We tested intergenic and intronic miRNAs (miR-10d, miR-21, miR-27a, miR-126a, miR-126b, miR-138, miR-140, miR-144, miR-196a1, miR-196a2, miR-196a2b [miR-196c], miR-196b, miR-196b1b [miR-196d], miR-199, miR-214, miR-200, and miR-222) in whole mounts and some of these in histological sections. Results showed that pri-miRNA ISH provides an attractive and cost-effective tool to study miRNA expression by ISH. We use this method to show that miR-126a and miR-126b are transcribed in the caudal vasculature in the pattern of their neighboring gene ci116 or host gene egfl7, respectively, and that the chondrocyte miRNA mir-140 lies downstream of Sox9 in development of the craniofacial skeleton.
PMCID: PMC3065723  PMID: 21288128
23.  MicroRNA-221 and microRNA-222 regulate gastric carcinoma cell proliferation and radioresistance by targeting PTEN 
BMC Cancer  2010;10:367.
MicroRNAs (miRNAs) can function as either oncogenes or tumor suppressor genes via regulation of cell proliferation and/or apoptosis. MiR-221 and miR-222 were discovered to induce cell growth and cell cycle progression via direct targeting of p27 and p57 in various human malignancies. However, the roles of miR-221 and miR-222 have not been reported in human gastric cancer. In this study, we examined the impact of miR-221 and miR-222 on human gastric cancer cells, and identified target genes for miR-221 and miR-222 that might mediate their biology.
The human gastric cancer cell line SGC7901 was transfected with AS-miR-221/222 or transduced with pMSCV-miR-221/222 to knockdown or restore expression of miR-221 and miR-222, respectively. The effects of miR-221 and miR-222 were then assessed by cell viability, cell cycle analysis, apoptosis, transwell, and clonogenic assay. Potential target genes were identified by Western blot and luciferase reporter assay.
Upregulation of miR-221 and miR-222 induced the malignant phenotype of SGC7901 cells, whereas knockdown of miR-221 and miR-222 reversed this phenotype via induction of PTEN expression. In addition, knockdonwn of miR-221 and miR-222 inhibited cell growth and invasion and increased the radiosensitivity of SGC7901 cells. Notably, the seed sequence of miR-221 and miR-222 matched the 3'UTR of PTEN, and introducing a PTEN cDNA without the 3'UTR into SGC7901 cells abrogated the miR-221 and miR-222-induced malignant phenotype. PTEN-3'UTR luciferase reporter assay confirmed PTEN as a direct target of miR-221 and miR-222.
These results demonstrate that miR-221 and miR-222 regulate radiosensitivity, and cell growth and invasion of SGC7901 cells, possibly via direct modulation of PTEN expression. Our study suggests that inhibition of miR-221 and miR-222 might form a novel therapeutic strategy for human gastric cancer.
PMCID: PMC2914702  PMID: 20618998
24.  Drosha Promotes Splicing of a Pre-microRNA-like Alternative Exon 
PLoS Genetics  2014;10(5):e1004312.
The ribonuclease III enzyme Drosha has a central role in the biogenesis of microRNA (miRNA) by binding and cleaving hairpin structures in primary RNA transcripts into precursor miRNAs (pre-miRNAs). Many miRNA genes are located within protein-coding host genes and cleaved by Drosha in a manner that is coincident with splicing of introns by the spliceosome. The close proximity of splicing and pre-miRNA biogenesis suggests a potential for co-regulation of miRNA and host gene expression, though this relationship is not completely understood. Here, we describe a cleavage-independent role for Drosha in the splicing of an exon that has a predicted hairpin structure resembling a Drosha substrate. We find that Drosha can cleave the alternatively spliced exon 5 of the eIF4H gene into a pre-miRNA both in vitro and in cells. However, the primary role of Drosha in eIF4H gene expression is to promote the splicing of exon 5. Drosha binds to the exon and enhances splicing in a manner that depends on RNA structure but not on cleavage by Drosha. We conclude that Drosha can function like a splicing enhancer and promote exon inclusion. Our results reveal a new mechanism of alternative splicing regulation involving a cleavage-independent role for Drosha in splicing.
Author Summary
MicroRNAs (miRNAs) are short non-coding RNAs that function in gene silencing and are produced by cleavage from a larger primary RNA transcript through a reaction that is carried out by the Microprocessor. Primary miRNA transcripts are often located within the introns of genes. Thus, both the Microprocessor and the spliceosome, which is responsible for pre-mRNA splicing, interact with the same sequences, though little is known about how these two processes influence each other. In this study, we discovered that the alternatively spliced eIF4H exon 5 is predicted to form an RNA hairpin that resembles a Microprocessor substrate. We found that the Microprocessor can bind and cleave exon 5, which precludes inclusion of the exon in the mRNA. However, we find that Drosha, a component of the Microprocessor, primarily functions to enhance exon 5 splicing both in vitro and in cells, rather than to cleave the RNA. Our results suggest that the Microprocessor has a role in splicing that is distinct from its role in miRNA biogenesis. This Microprocessor activity represents a new function for the complex that may be an important mechanism for regulating alternative splicing.
PMCID: PMC4006729  PMID: 24786770
25.  miR-198 Inhibits HIV-1 Gene Expression and Replication in Monocytes and Its Mechanism of Action Appears To Involve Repression of Cyclin T1 
PLoS Pathogens  2009;5(1):e1000263.
Cyclin T1 is a regulatory subunit of a general RNA polymerase II elongation factor known as P-TEFb. Cyclin T1 is also required for Tat transactivation of HIV-1 LTR-directed gene expression. Translation of Cyclin T1 mRNA has been shown to be repressed in human monocytes, and this repression is relieved when cells differentiate to macrophages. We identified miR-198 as a microRNA (miRNA) that is strongly down-regulated when monocytes are induced to differentiate. Ectopic expression of miR-198 in tissue culture cells reduced Cyclin T1 protein expression, and plasmid reporter assays verified miR-198 target sequences in the 3′ untranslated region (3′UTR) of Cyclin T1 mRNA. Cyclin T1 protein levels increased when an inhibitor of miR-198 was transfected into primary monocytes, and overexpression of miR-198 in primary monocytes repressed the normal up-regulation of Cyclin T1 during differentiation. Expression of an HIV-1 proviral plasmid and HIV-1 replication were repressed in a monocytic cell line upon overexpression of miR-198. Our data indicate that miR-198 functions to restrict HIV-1 replication in monocytes, and its mechanism of action appears to involve repression of Cyclin T1 expression.
Author Summary
Monocytes do not support HIV-1 replication, in part because they do not express adequate levels of essential cellular cofactors that mediate steps in the viral replication cycle. Monocytes become permissive for viral replication upon differentiation to macrophages, indicating that cellular cofactors are induced during the differentiation process. One such cofactor is Cyclin T1, which is not expressed in monocytes and is expressed at high levels following macrophage differentiation. Cyclin T1 functions to greatly stimulate the amount of HIV-1 produced in the infected cell. We identified a microRNA (miRNA) named miR-198 that represses the expression of Cyclin T1 in monocytes. miRNAs block expression of proteins by binding to messenger RNAs and preventing their translation by ribosomes. The expression levels of miR-198 are greatly reduced in macrophages, and this appears to allow translation of Cyclin T1 mRNA and expression of Cyclin T1 protein. Our study indicates that this miRNA restricts HIV-1 replication in monocytes. We think that it is possible, if not likely, that additional miRNAs in monocytes also restrict HIV-1 replication by repressing other essential cellular cofactors.
PMCID: PMC2607557  PMID: 19148268

Results 1-25 (940903)