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1.  Seq and CLIP through the miRNA world 
Genome Biology  2014;15(1):202.
High-throughput sequencing of RNAs crosslinked to Argonaute proteins reveals not only a multitude of atypical miRNA binding sites but also of miRNA targets with atypical functions, and can be used to infer quantitative models of miRNA-target interaction strength.
doi:10.1186/gb4151
PMCID: PMC4053862  PMID: 24460822
2.  PAR-CLIP (Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation): a Step-By-Step Protocol to the Transcriptome-Wide Identification of Binding Sites of RNA-Binding Proteins 
Methods in enzymology  2014;539:113-161.
We recently developed a protocol for the transcriptome-wide isolation of RNA recognition elements readily applicable to any protein or ribonucleoprotein complex directly contacting RNA (including RNA helicases, polymerases, or nucleases) expressed in cell culture models either naturally or ectopically (Hafner et al., 2010).
Briefly, immunoprecipitation of the RNA-binding protein of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. In the course of lysate preparation and immunoprecipitation, the mRNAs are partially degraded using Ribonu-clease T1. The isolated crosslinked RNA fragments are converted into a cDNA library and deep-sequenced using Solexa technology (see Explanatory Chapter: Next Generation Sequencing). By introducing photoreactive nucleosides that generate characteristic sequence changes upon crosslinking (see below), our protocol allows one to separate RNA segments bound by the protein of interest from the background un-crosslinked RNAs.
doi:10.1016/B978-0-12-420120-0.00008-6
PMCID: PMC4180672  PMID: 24581442
3.  Embryonic stem cell-specific microRNAs contribute to pluripotency by inhibiting regulators of multiple differentiation pathways 
Nucleic Acids Research  2014;42(14):9313-9326.
The findings that microRNAs (miRNAs) are essential for early development in many species and that embryonic miRNAs can reprogram somatic cells into induced pluripotent stem cells suggest that these miRNAs act directly on transcriptional and chromatin regulators of pluripotency. To elucidate the transcription regulatory networks immediately downstream of embryonic miRNAs, we extended the motif activity response analysis approach that infers the regulatory impact of both transcription factors (TFs) and miRNAs from genome-wide expression states. Applying this approach to multiple experimental data sets generated from mouse embryonic stem cells (ESCs) that did or did not express miRNAs of the ESC-specific miR-290-295 cluster, we identified multiple TFs that are direct miRNA targets, some of which are known to be active during cell differentiation. Our results provide new insights into the transcription regulatory network downstream of ESC-specific miRNAs, indicating that these miRNAs act on cell cycle and chromatin regulators at several levels and downregulate TFs that are involved in the innate immune response.
doi:10.1093/nar/gku544
PMCID: PMC4132708  PMID: 25030899
4.  Structural and functional implications of the QUA2 domain on RNA recognition by GLD-1 
Nucleic Acids Research  2014;42(12):8092-8105.
The STAR family comprises ribonucleic acid (RNA)-binding proteins that play key roles in RNA-regulatory processes. RNA recognition is achieved by a KH domain with an additional α-helix (QUA2) that seems to extend the RNA-binding surface to six nucleotides for SF1 (Homo sapiens) and seven nucleotides for GLD-1 (Caenorhabditis elegans). To understand the structural basis of this probable difference in specificity, we determined the solution structure of GLD-1 KH-QUA2 with the complete consensus sequence identified in the tra-2 gene. Compared to SF1, the GLD-1 KH-QUA2 interface adopts a different conformation resulting indeed in an additional sequence-specific binding pocket for a uracil at the 5′end. The functional relevance of this binding pocket is emphasized by our bioinformatics analysis showing that GLD-1 binding sites with this 5′end uracil are more predictive for the functional response of the messenger RNAs to gld-1 knockout. We further reveal the importance of the KH-QUA2 interface in vitro and that its alteration in vivo affects the level of translational repression dependent on the sequence of the GLD-1 binding motif. In conclusion, we demonstrate that the QUA2 domain distinguishes GLD-1 from other members of the STAR family and contributes more generally to the modulation of RNA-binding affinity and specificity of KH domain containing proteins.
doi:10.1093/nar/gku445
PMCID: PMC4081071  PMID: 24838563
5.  Fifteen years SIB Swiss Institute of Bioinformatics: life science databases, tools and support 
Nucleic Acids Research  2014;42(Web Server issue):W436-W441.
The SIB Swiss Institute of Bioinformatics (www.isb-sib.ch) was created in 1998 as an institution to foster excellence in bioinformatics. It is renowned worldwide for its databases and software tools, such as UniProtKB/Swiss-Prot, PROSITE, SWISS-MODEL, STRING, etc, that are all accessible on ExPASy.org, SIB's Bioinformatics Resource Portal. This article provides an overview of the scientific and training resources SIB has consistently been offering to the life science community for more than 15 years.
doi:10.1093/nar/gku380
PMCID: PMC4086091  PMID: 24792157
6.  MicroRNA-7a regulates pancreatic β cell function 
The Journal of Clinical Investigation  2014;124(6):2722-2735.
Dysfunctional microRNA (miRNA) networks contribute to inappropriate responses following pathological stress and are the underlying cause of several disease conditions. In pancreatic β cells, miRNAs have been largely unstudied and little is known about how specific miRNAs regulate glucose-stimulated insulin secretion (GSIS) or impact the adaptation of β cell function to metabolic stress. In this study, we determined that miR-7 is a negative regulator of GSIS in β cells. Using Mir7a2 deficient mice, we revealed that miR-7a2 regulates β cell function by directly regulating genes that control late stages of insulin granule fusion with the plasma membrane and ternary SNARE complex activity. Transgenic mice overexpressing miR-7a in β cells developed diabetes due to impaired insulin secretion and β cell dedifferentiation. Interestingly, perturbation of miR-7a expression in β cells did not affect proliferation and apoptosis, indicating that miR-7 is dispensable for the maintenance of endocrine β cell mass. Furthermore, we found that miR-7a levels are decreased in obese/diabetic mouse models and human islets from obese and moderately diabetic individuals with compensated β cell function. Our results reveal an interconnecting miR-7 genomic circuit that regulates insulin granule exocytosis in pancreatic β cells and support a role for miR-7 in the adaptation of pancreatic β cell function in obesity and type 2 diabetes.
doi:10.1172/JCI73066
PMCID: PMC4038573  PMID: 24789908
7.  Argonaute2 Mediates Compensatory Expansion of the Pancreatic β Cell 
Cell Metabolism  2014;19(1):122-134.
Summary
Pancreatic β cells adapt to compensate for increased metabolic demand during insulin resistance. Although the microRNA pathway has an essential role in β cell proliferation, the extent of its contribution is unclear. Here, we report that miR-184 is silenced in the pancreatic islets of insulin-resistant mouse models and type 2 diabetic human subjects. Reduction of miR-184 promotes the expression of its target Argonaute2 (Ago2), a component of the microRNA-induced silencing complex. Moreover, restoration of miR-184 in leptin-deficient ob/ob mice decreased Ago2 and prevented compensatory β cell expansion. Loss of Ago2 during insulin resistance blocked β cell growth and relieved the regulation of miR-375-targeted genes, including the growth suppressor Cadm1. Lastly, administration of a ketogenic diet to ob/ob mice rescued insulin sensitivity and miR-184 expression and restored Ago2 and β cell mass. This study identifies the targeting of Ago2 by miR-184 as an essential component of the compensatory response to regulate proliferation according to insulin sensitivity.
Graphical Abstract
Highlights
•Silencing of miR-184 during insulin resistance promotes its target Ago2•Loss of Ago2 during insulin resistance blocks pancreatic β cell proliferation•Ago2 mediates the suppression of Cadm1 by miR-375 in the β cell•Administration of the ketogenic diet to ob/ob mice rescues miR-184 in islets
Tattikota et al. find that as β cells adapt to increased metabolic demand during insulin resistance in obesity, miR-184 is silenced to alleviate repression of its target Argonaute2, a component of the microRNA-induced silencing complex. Argonaute2 promotes compensatory β cell proliferation via miR-375 and its target genes, including the growth suppressor Cadm1.
doi:10.1016/j.cmet.2013.11.015
PMCID: PMC3945818  PMID: 24361012
8.  Timescales and bottlenecks in miRNA-dependent gene regulation 
Application of a kinetic model of miRNA-mediated gene regulation to experimental data sets shows that the timescale of regulation is slower than previously assumed, due to bottlenecks imposed by miRNA turnover in the RNA-induced silencing complex and by slow protein decay.
A mathematical model links the dynamics of miRNA expression and loading into the Argonaute protein to the dynamics of miRNA targets.Loading of miRNAs into Argonaute and the slow decay of proteins impose two bottlenecks on the speed of miRNA-mediated regulation.Accelerated miRNA turnover is necessary for regulating target expression on the timescale of a day.
MiRNAs are post-transcriptional regulators that contribute to the establishment and maintenance of gene expression patterns. Although their biogenesis and decay appear to be under complex control, the implications of miRNA expression dynamics for the processes that they regulate are not well understood. We derived a mathematical model of miRNA-mediated gene regulation, inferred its parameters from experimental data sets, and found that the model describes well time-dependent changes in mRNA, protein and ribosome density levels measured upon miRNA transfection and induction. The inferred parameters indicate that the timescale of miRNA-dependent regulation is slower than initially thought. Delays in miRNA loading into Argonaute proteins and the slow decay of proteins relative to mRNAs can explain the typically small changes in protein levels observed upon miRNA transfection. For miRNAs to regulate protein expression on the timescale of a day, as miRNAs involved in cell-cycle regulation do, accelerated miRNA turnover is necessary.
doi:10.1038/msb.2013.68
PMCID: PMC3882800  PMID: 24301800
gene expression regulation; kinetics; miRNAs; modeling; protein turnover
9.  Dicer partners expand the repertoire of miRNA targets 
Genome Biology  2012;13(11):179.
Processing of pre-miRNAs by Dicer is regulated by its dsRNA-binding protein partner, and leads to the generation of alternative miRNA forms with distinct target sets.
doi:10.1186/gb-2012-13-11-179
PMCID: PMC3580490  PMID: 23194401
10.  Insights into snoRNA biogenesis and processing from PAR-CLIP of snoRNA core proteins and small RNA sequencing 
Genome Biology  2013;14(5):R45.
Background
In recent years, a variety of small RNAs derived from other RNAs with well-known functions such as tRNAs and snoRNAs, have been identified. The functional relevance of these RNAs is largely unknown. To gain insight into the complexity of snoRNA processing and the functional relevance of snoRNA-derived small RNAs, we sequence long and short RNAs, small RNAs that co-precipitate with the Argonaute 2 protein and RNA fragments obtained in photoreactive nucleotide-enhanced crosslinking and immunoprecipitation (PAR-CLIP) of core snoRNA-associated proteins.
Results
Analysis of these data sets reveals that many loci in the human genome reproducibly give rise to C/D box-like snoRNAs, whose expression and evolutionary conservation are typically less pronounced relative to the snoRNAs that are currently cataloged. We further find that virtually all C/D box snoRNAs are specifically processed inside the regions of terminal complementarity, retaining in the mature form only 4-5 nucleotides upstream of the C box and 2-5 nucleotides downstream of the D box. Sequencing of the total and Argonaute 2-associated populations of small RNAs reveals that despite their cellular abundance, C/D box-derived small RNAs are not efficiently incorporated into the Ago2 protein.
Conclusions
We conclude that the human genome encodes a large number of snoRNAs that are processed along the canonical pathway and expressed at relatively low levels. Generation of snoRNA-derived processing products with alternative, particularly miRNA-like, functions appears to be uncommon.
doi:10.1186/gb-2013-14-5-r45
PMCID: PMC4053766  PMID: 23706177
11.  Kaposi's Sarcoma Herpesvirus microRNAs Target Caspase 3 and Regulate Apoptosis 
PLoS Pathogens  2011;7(12):e1002405.
Kaposi's sarcoma herpesvirus (KSHV) encodes a cluster of twelve micro (mi)RNAs, which are abundantly expressed during both latent and lytic infection. Previous studies reported that KSHV is able to inhibit apoptosis during latent infection; we thus tested the involvement of viral miRNAs in this process. We found that both HEK293 epithelial cells and DG75 cells stably expressing KSHV miRNAs were protected from apoptosis. Potential cellular targets that were significantly down-regulated upon KSHV miRNAs expression were identified by microarray profiling. Among them, we validated by luciferase reporter assays, quantitative PCR and western blotting caspase 3 (Casp3), a critical factor for the control of apoptosis. Using site-directed mutagenesis, we found that three KSHV miRNAs, miR-K12-1, 3 and 4-3p, were responsible for the targeting of Casp3. Specific inhibition of these miRNAs in KSHV-infected cells resulted in increased expression levels of endogenous Casp3 and enhanced apoptosis. Altogether, our results suggest that KSHV miRNAs directly participate in the previously reported inhibition of apoptosis by the virus, and are thus likely to play a role in KSHV-induced oncogenesis.
Author Summary
MiRNAs are small, non-coding RNAs that regulate gene expression post-transcriptionally via binding to complementary sites in target mRNAs. This evolutionary conserved regulatory system is present in most eukaryotes, and it has recently been shown that certain viruses have evolved to express their own miRNAs. Due to their non-immunogenic nature, viral miRNAs represent an efficient tool for the virus to control its environment. Here we show that KSHV miRNAs are involved in the control of apoptosis both when expressed in stable cell lines and in the context of viral infection. Using a microarray based approach we identified putative cellular targets, among which the effector caspase 3 is targeted by three of the viral miRNAs. Finally, we showed that blocking these miRNAs in infected cells resulted both in increased Casp3 levels and a higher apoptosis rate. These findings indicate that miRNAs of viral origin are key players in cell death inhibition by KSHV.
doi:10.1371/journal.ppat.1002405
PMCID: PMC3234232  PMID: 22174674
12.  Deciphering the role of RNA-binding proteins in the post-transcriptional control of gene expression 
Briefings in Functional Genomics  2010;9(5-6):391-404.
Eukaryotic cells express a large variety of ribonucleic acid-(RNA)-binding proteins (RBPs) with diverse affinity and specificity towards target RNAs that play a crucial role in almost every aspect of RNA metabolism. In addition, specific domains in RBPs impart catalytic activity or mediate protein–protein interactions, making RBPs versatile regulators of gene expression. In this review, we elaborate on recent experimental and computational approaches that have increased our understanding of RNA–protein interactions and their role in cellular function. We review aspects of gene expression that are modulated post-transcriptionally by RBPs, namely the stability of polymerase II-derived mRNA transcripts and their rate of translation into proteins. We further highlight the extensive regulatory networks of RBPs that implement a combinatorial control of gene expression. Taking cues from the recent development in the field, we argue that understanding spatio-temporal RNA–protein association on a transcriptome level will provide invaluable and unexpected insights into the regulatory codes that define growth, differentiation and disease.
doi:10.1093/bfgp/elq028
PMCID: PMC3080770  PMID: 21127008
RNA-binding proteins; RNA-binding domains; RBP–RNA interaction; RBP regulatory networks; RBP target identification
13.  The snoRNA MBII-52 (SNORD 115) is processed into smaller RNAs and regulates alternative splicing 
Human Molecular Genetics  2010;19(7):1153-1164.
The loss of HBII-52 and related C/D box small nucleolar RNA (snoRNA) expression units have been implicated as a cause for the Prader–Willi syndrome (PWS). We recently found that the C/D box snoRNA HBII-52 changes the alternative splicing of the serotonin receptor 2C pre-mRNA, which is different from the traditional C/D box snoRNA function in non-mRNA methylation. Using bioinformatic predictions and experimental verification, we identified five pre-mRNAs (DPM2, TAF1, RALGPS1, PBRM1 and CRHR1) containing alternative exons that are regulated by MBII-52, the mouse homolog of HBII-52. Analysis of a single member of the MBII-52 cluster of snoRNAs by RNase protection and northern blot analysis shows that the MBII-52 expressing unit generates shorter RNAs that originate from the full-length MBII-52 snoRNA through additional processing steps. These novel RNAs associate with hnRNPs and not with proteins associated with canonical C/D box snoRNAs. Our data indicate that not a traditional C/D box snoRNA MBII-52, but a processed version lacking the snoRNA stem is the predominant MBII-52 RNA missing in PWS. This processed snoRNA functions in alternative splice-site selection. Its substitution could be a therapeutic principle for PWS.
doi:10.1093/hmg/ddp585
PMCID: PMC2838533  PMID: 20053671
14.  microRNA activity is suppressed in mouse oocytes 
Current biology : CB  2010;20(3):265-270.
Summary
MicroRNAs (miRNAs) are small endogenous RNAs, which typically imperfectly base-pair with 3′UTRs and mediate translational repression and mRNA degradation. Dicer, an RNase III generating small RNAs in the miRNA and RNAi pathways, is essential for meiotic maturation of mouse oocytes. We found that 3′UTRs of transcripts up-regulated in Dicer1−/− oocytes are not enriched in miRNA binding sites implicating a weak impact of miRNAs on the maternal transcriptome. Therefore, we tested the ability of endogenous miRNAs to mediate RNA-like cleavage or translational repression of reporter mRNAs. In contrast to somatic cells, endogenous miRNAs in fully-grown GV oocytes poorly repressed translation of mRNA reporters whereas their RNAi-like activity was much less affected. In addition, reporter mRNA carrying let-7-binding sites failed to localize to P-body-like structures in oocytes. Our data suggest that normal miRNA function is down-regulated during oocyte development and this idea is further supported by normal meiotic maturation of oocytes lacking Dgcr8, which is required for the miRNA but not the RNAi pathway [Suh et al.]. We propose that suppression of miRNA function during oocyte growth is an early event in reprogramming gene expression during the transition of a differentiated oocyte into pluripotent blastomeres of the embryo.
doi:10.1016/j.cub.2009.12.042
PMCID: PMC2824427  PMID: 20116252
miRNA; endo-siRNA; P-body; maternal mRNA; oocyte; mRNA stability; mRNA degradation; translational arrest
15.  Conserved generation of short products at piRNA loci 
BMC Genomics  2011;12:46.
Background
The piRNA pathway operates in animal germ lines to ensure genome integrity through retrotransposon silencing. The Piwi protein-associated small RNAs (piRNAs) guide Piwi proteins to retrotransposon transcripts, which are degraded and thereby post-transcriptionally silenced through a ping-pong amplification process. Cleavage of the retrotransposon transcript defines at the same time the 5' end of a secondary piRNA that will in turn guide a Piwi protein to a primary piRNA precursor, thereby amplifying primary piRNAs. Although several studies provided evidence that this mechanism is conserved among metazoa, how the process is initiated and what enzymatic activities are responsible for generating the primary and secondary piRNAs are not entirely clear.
Results
Here we analyzed small RNAs from three mammalian species, seeking to gain further insight into the mechanisms responsible for the piRNA amplification loop. We found that in all these species piRNA-directed targeting is accompanied by the generation of short sequences that have a very precisely defined length, 19 nucleotides, and a specific spatial relationship with the guide piRNAs.
Conclusions
This suggests that the processing of the 5' product of piRNA-guided cleavage occurs while the piRNA target is engaged by the Piwi protein. Although they are not stabilized through methylation of their 3' ends, the 19-mers are abundant not only in testes lysates but also in immunoprecipitates of Miwi and Mili proteins. They will enable more accurate identification of piRNA loci in deep sequencing data sets.
doi:10.1186/1471-2164-12-46
PMCID: PMC3037900  PMID: 21247452
16.  CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins 
Nucleic Acids Research  2010;39(Database issue):D245-D252.
The stability, localization and translation rate of mRNAs are regulated by a multitude of RNA-binding proteins (RBPs) that find their targets directly or with the help of guide RNAs. Among the experimental methods for mapping RBP binding sites, cross-linking and immunoprecipitation (CLIP) coupled with deep sequencing provides transcriptome-wide coverage as well as high resolution. However, partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, we have developed the CLIPZ database and analysis environment. Binding site data for RBPs such as Argonaute 1-4, Insulin-like growth factor II mRNA-binding protein 1-3, TNRC6 proteins A-C, Pumilio 2, Quaking and Polypyrimidine tract binding protein can be visualized at the level of the genome and of individual transcripts. Individual users can upload their own sequence data sets while being able to limit the access to these data to specific users, and analyses of the public and private data sets can be performed interactively. CLIPZ, available at http://www.clipz.unibas.ch, aims to provide an open access repository of information for post-transcriptional regulatory elements.
doi:10.1093/nar/gkq940
PMCID: PMC3013791  PMID: 21087992
17.  Analysis of in situ pre-mRNA targets of human splicing factor SF1 reveals a function in alternative splicing 
Nucleic Acids Research  2010;39(5):1868-1879.
The conserved pre-mRNA splicing factor SF1 is implicated in 3′ splice site recognition by binding directly to the intron branch site. However, because SF1 is not essential for constitutive splicing, its role in pre-mRNA processing has remained mysterious. Here, we used crosslinking and immunoprecipitation (CLIP) to analyze short RNAs directly bound by human SF1 in vivo. SF1 bound mainly pre-mRNAs, with 77% of target sites in introns. Binding to target RNAs in vitro was dependent on the newly defined SF1 binding motif ACUNAC, strongly resembling human branch sites. Surprisingly, the majority of SF1 binding sites did not map to the expected position near 3′ splice sites. Instead, target sites were distributed throughout introns, and a smaller but significant fraction occurred in exons within coding and untranslated regions. These data suggest a more complex role for SF1 in splicing regulation. Indeed, SF1 silencing affected alternative splicing of endogenous transcripts, establishing a previously unexpected role for SF1 and branch site-like sequences in splice site selection.
doi:10.1093/nar/gkq1042
PMCID: PMC3061054  PMID: 21062807
18.  Expression proteomics of UPF1 knockdown in HeLa cells reveals autoregulation of hnRNP A2/B1 mediated by alternative splicing resulting in nonsense-mediated mRNA decay 
BMC Genomics  2010;11:565.
Background
In addition to acting as an RNA quality control pathway, nonsense-mediated mRNA decay (NMD) plays roles in regulating normal gene expression. In particular, the extent to which alternative splicing is coupled to NMD and the roles of NMD in regulating uORF containing transcripts have been a matter of debate.
Results
In order to achieve a greater understanding of NMD regulated gene expression we used 2D-DiGE proteomics technology to examine the changes in protein expression induced in HeLa cells by UPF1 knockdown. QPCR based validation of the corresponding mRNAs, in response to both UPF1 knockdown and cycloheximide treatment, identified 17 bona fide NMD targets. Most of these were associated with bioinformatically predicted NMD activating features, predominantly upstream open reading frames (uORFs). Strikingly, however, the majority of transcripts up-regulated by UPF1 knockdown were either insensitive to, or even down-regulated by, cycloheximide treatment. Furthermore, the mRNA abundance of several down-regulated proteins failed to change upon UPF1 knockdown, indicating that UPF1's role in regulating mRNA and protein abundance is more complex than previously appreciated. Among the bona fide NMD targets, we identified a highly conserved AS-NMD event within the 3' UTR of the HNRNPA2B1 gene. Overexpression of GFP tagged hnRNP A2 resulted in a decrease in endogenous hnRNP A2 and B1 mRNA with a concurrent increase in the NMD sensitive isoforms.
Conclusions
Despite the large number of changes in protein expression upon UPF1 knockdown, a relatively small fraction of them can be directly attributed to the action of NMD on the corresponding mRNA. From amongst these we have identified a conserved AS-NMD event within HNRNPA2B1 that appears to mediate autoregulation of HNRNPA2B1 expression levels.
doi:10.1186/1471-2164-11-565
PMCID: PMC3091714  PMID: 20946641
19.  PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins 
RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18
doi:10.3791/2034
PMCID: PMC3156069  PMID: 20644507
20.  Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP 
Cell  2010;141(1):129-141.
Summary
RNA transcripts are subject to post-transcriptional gene regulation involving hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) expressed in a cell-type dependent fashion. We developed a cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs. The crosslinked sites are revealed by thymidine to cytidine transitions in the cDNAs prepared from immunopurified RNPs of 4-thiouridine-treated cells. We determined the binding sites and regulatory consequences for several intensely studied RBPs and miRNPs, including PUM2, QKI, IGF2BP1-3, AGO/EIF2C1-4 and TNRC6A-C. Our study revealed that these factors bind thousands of sites containing defined sequence motifs and have distinct preferences for exonic versus intronic or coding versus untranslated transcript regions. The precise mapping of binding sites across the transcriptome will be critical to the interpretation of the rapidly emerging data on genetic variation between individuals and how these variations contribute to complex genetic diseases.
doi:10.1016/j.cell.2010.03.009
PMCID: PMC2861495  PMID: 20371350
21.  miRNA in situ hybridization in mammalian tissues fixed with formaldehyde and EDC 
Nature methods  2009;6(2):139-141.
MicroRNAs (miRNAs) are small regulatory RNAs with many biological functions and disease associations. We showed that in situ hybridization (ISH) using conventional formaldehyde fixation results in significant miRNA loss from mouse tissue sections, which can be prevented by fixation with 1–ethyl–3–(3–dimethylaminopropyl) carbodiimide (EDC) that irreversibly immobilizes the miRNA at its 5' phosphate. We determined optimal hybridization parameters for 130 locked nucleic acid (LNA) probes by recording nucleic acid melting temperature during ISH.
doi:10.1038/nmeth.1294
PMCID: PMC2838186  PMID: 19137005
immunohistochemistry; heart; liver; brain; dendrites; methanal; paraformaldehyde; water–soluble carbodiimide; phosphoramidate; crosslink; non–coding RNA; small RNA; locked nucleic acid probe
22.  Expression and Processing of a Small Nucleolar RNA from the Epstein-Barr Virus Genome 
PLoS Pathogens  2009;5(8):e1000547.
Small nucleolar RNAs (snoRNAs) are localized within the nucleolus, a sub-nuclear compartment, in which they guide ribosomal or spliceosomal RNA modifications, respectively. Up until now, snoRNAs have only been identified in eukaryal and archaeal genomes, but are notably absent in bacteria. By screening B lymphocytes for expression of non-coding RNAs (ncRNAs) induced by the Epstein-Barr virus (EBV), we here report, for the first time, the identification of a snoRNA gene within a viral genome, designated as v-snoRNA1. This genetic element displays all hallmark sequence motifs of a canonical C/D box snoRNA, namely C/C′- as well as D/D′-boxes. The nucleolar localization of v-snoRNA1 was verified by in situ hybridisation of EBV-infected cells. We also confirmed binding of the three canonical snoRNA proteins, fibrillarin, Nop56 and Nop58, to v-snoRNA1. The C-box motif of v-snoRNA1 was shown to be crucial for the stability of the viral snoRNA; its selective deletion in the viral genome led to a complete down-regulation of v-snoRNA1 expression levels within EBV-infected B cells. We further provide evidence that v-snoRNA1 might serve as a miRNA-like precursor, which is processed into 24 nt sized RNA species, designated as v-snoRNA124pp. A potential target site of v-snoRNA124pp was identified within the 3′-UTR of BALF5 mRNA which encodes the viral DNA polymerase. V-snoRNA1 was found to be expressed in all investigated EBV-positive cell lines, including lymphoblastoid cell lines (LCL). Interestingly, induction of the lytic cycle markedly up-regulated expression levels of v-snoRNA1 up to 30-fold. By a computational approach, we identified a v-snoRNA1 homolog in the rhesus lymphocryptovirus genome. This evolutionary conservation suggests an important role of v-snoRNA1 during γ-herpesvirus infection.
Author Summary
Epstein-Barr virus (EBV) infects about 90% of people worldwide and is associated with different types of cancer. So far, only two large virus-encoded non-coding RNAs (EBER1 and EBER2) and 25 microRNAs (miRNAs) have been identified in the EBV genome. In this study, we report identification of the first member of another abundant non-coding RNA class, a small nucleolar RNA (snoRNA), designated as v-snoRNA1. We show that v-snoRNA1 is located in the nucleolus and interacts with the same proteins as reported for canonical eukaryal snoRNAs. Its biological function is consistent with its high conservation in a distantly related simian herpesvirus genome. Interestingly, v-snoRNA1 might serve as a miRNA-like precursor, which is processed into a 24 nt sized RNA species, designated as v-snoRNA124pp. The viral DNA polymerase BALF5 was identified as a potential target for v-snoRNA124pp. Taken together, these experiments strengthen the crucial function of v-snoRNA1 in EBV infection.
doi:10.1371/journal.ppat.1000547
PMCID: PMC2718842  PMID: 19680535
23.  MirZ: an integrated microRNA expression atlas and target prediction resource 
Nucleic Acids Research  2009;37(Web Server issue):W266-W272.
MicroRNAs (miRNAs) are short RNAs that act as guides for the degradation and translational repression of protein-coding mRNAs. A large body of work showed that miRNAs are involved in the regulation of a broad range of biological functions, from development to cardiac and immune system function, to metabolism, to cancer. For most of the over 500 miRNAs that are encoded in the human genome the functions still remain to be uncovered. Identifying miRNAs whose expression changes between cell types or between normal and pathological conditions is an important step towards characterizing their function as is the prediction of mRNAs that could be targeted by these miRNAs. To provide the community the possibility of exploring interactively miRNA expression patterns and the candidate targets of miRNAs in an integrated environment, we developed the MirZ web server, which is accessible at www.mirz.unibas.ch. The server provides experimental and computational biologists with statistical analysis and data mining tools operating on up-to-date databases of sequencing-based miRNA expression profiles and of predicted miRNA target sites in species ranging from Caenorhabditis elegans to Homo sapiens.
doi:10.1093/nar/gkp412
PMCID: PMC2703880  PMID: 19468042
24.  A Mammalian microRNA Expression Atlas Based on Small RNA Library Sequencing 
Cell  2007;129(7):1401-1414.
Summary
MicroRNAs (miRNAs) are small non-coding regulatory RNAs that reduce stability and/or translation of fully or partially sequence-complementary target mRNAs. In order to identify miRNAs and to assess their expression patterns, we sequenced over 250 small RNA libraries from 26 different organ systems and cell types of human and rodents, enriched in neuronal as well as normal and malignant hematopoietic cells and tissues. We present expression profiles derived from clone count data and provide novel computational tools for their analysis. Unexpectedly, a relatively small set of miRNAs, many of which are ubiquitously expressed, account for most of the difference in miRNA profiles between cell lineages and tissues. This broad survey also provides detailed and accurate information about mature sequences, precursors, genome locations, maturation processes, inferred transcriptional units and conservation patterns. We also propose a subclassification scheme for miRNAs for assisting future experimental and computational functional analyses.
doi:10.1016/j.cell.2007.04.040
PMCID: PMC2681231  PMID: 17604727
25.  Comparative Analysis of mRNA Targets for Human PUF-Family Proteins Suggests Extensive Interaction with the miRNA Regulatory System 
PLoS ONE  2008;3(9):e3164.
Genome-wide identification of mRNAs regulated by RNA-binding proteins is crucial to uncover post-transcriptional gene regulatory systems. The conserved PUF family RNA-binding proteins repress gene expression post-transcriptionally by binding to sequence elements in 3′-UTRs of mRNAs. Despite their well-studied implications for development and neurogenesis in metazoa, the mammalian PUF family members are only poorly characterized and mRNA targets are largely unknown. We have systematically identified the mRNAs associated with the two human PUF proteins, PUM1 and PUM2, by the recovery of endogenously formed ribonucleoprotein complexes and the analysis of associated RNAs with DNA microarrays. A largely overlapping set comprised of hundreds of mRNAs were reproducibly associated with the paralogous PUM proteins, many of them encoding functionally related proteins. A characteristic PUF-binding motif was highly enriched among PUM bound messages and validated with RNA pull-down experiments. Moreover, PUF motifs as well as surrounding sequences exhibit higher conservation in PUM bound messages as opposed to transcripts that were not found to be associated, suggesting that PUM function may be modulated by other factors that bind conserved elements. Strikingly, we found that PUF motifs are enriched around predicted miRNA binding sites and that high-confidence miRNA binding sites are significantly enriched in the 3′-UTRs of experimentally determined PUM1 and PUM2 targets, strongly suggesting an interaction of human PUM proteins with the miRNA regulatory system. Our work suggests extensive connections between the RBP and miRNA post-transcriptional regulatory systems and provides a framework for deciphering the molecular mechanism by which PUF proteins regulate their target mRNAs.
doi:10.1371/journal.pone.0003164
PMCID: PMC2522278  PMID: 18776931

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