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1.  Ensemble Analysis of Primary miRNA Structure Reveals an Extensive Capacity to Deform near the Drosha Cleavage Site 
Biochemistry  2013;52(5):795-807.
Most non-coding RNAs function properly only when folded into complex 3D structures, but the experimental determination of these structures remains challenging. Understanding of primary miRNA maturation is currently limited by a lack of solved structures for non-processed forms of the RNA. SHAPE chemistry efficiently determines RNA secondary structural information with single-nucleotide resolution, providing constraints suitable for input into the MC-Pipeline software for refinement of 3D structure models. Here we combine these approaches to analyze three structurally diverse primary miRNAs, revealing deviations from canonical dsRNA structure in the stem adjacent to the Drosha cut site for all three. The necessity of these deformable sites for efficient processing is demonstrated through Drosha processing assays. The structure models generated herein support the hypothesis that deformable sequences spaced roughly once per turn of A-form helix, created by non-canonical structure elements, combine with the necessary single-stranded RNA:double-stranded RNA junction to define the correct Drosha cleavage site.
doi:10.1021/bi301452a
PMCID: PMC3565094  PMID: 23305493
SHAPE; MC-Pipeline; chemical modification; RNA structure; microRNA; Drosha
2.  Sensitive PCR-Based Quantitation of Cell-Free Circulating MicroRNAs 
Methods (San Diego, Calif.)  2012;58(2):144-150.
Cell-free microRNAs (miRNAs) that circulate in the blood are promising surrogate biomarkers of disease and physiological processes. The ease of quantifying specific miRNA species using made-to-order approaches based on Taq-polymerase has led to numerous studies that have identified changes in the abundance of circulating cell-free miRNA species that correlate with pathology or other events. The growing interest in developing miRNAs as blood biomarkers necessitates the careful consideration of the unique properties of such body fluids that can make the reproducible and quantitative assessment of RNA abundance challenging. For example, enzymes involved in the amplification and analysis of RNA can be affected by blood components that copurify with miRNA. Thus, if miRNAs are to be effectively utilized as biomarkers, it is important to establish standardized protocols for blood collection and miRNA analysis to ensure accurate quantitation. Here we outline several considerations, including the type of collection tube used in sampling, the influence of added anticoagulants and stabilizers, sample processing, enrichment of vesicular and other miRNA species, RNA extraction approaches and enzyme selection, that affect quantitation of miRNA isolated from plasma and should be considered in order to achieve reproducible, sensitive and accurate quantitation.
doi:10.1016/j.ymeth.2012.07.026
PMCID: PMC3508311  PMID: 22884953
3.  Induction of Antagonistic Soluble Decoy Receptor Tyrosine Kinases by Intronic PolyA Activation 
Molecular cell  2011;43(6):927-939.
SUMMARY
Alternative intronic polyadenylation (IPA) can generate truncated protein isoforms with significantly altered functions. Here, we describe 31 dominant-negative, secreted variant isoforms of receptor tyrosine kinases (RTKs) that are produced by activation of intronic poly(A) sites. We show that blocking U1-snRNP can activate IPA, indicating a larger role for U1-snRNP in RNA surveillance. Moreover, we report the development of an antisense-based method to effectively and specifically activate expression of individual soluble decoy RTKs (sdRTKs) to alter signaling, with potential therapeutic implications. In particular, a quantitative switch from signal transducing full-length vascular endothelial growth factor receptor-2 (VEGFR2/KDR) to a dominant-negative sKDR results in a strong anti-angiogenic effect both on directly targeted cells and on naïve cells exposed to conditioned media, suggesting a role for this approach in interfering with angiogenic paracrine and autocrine loops.
doi:10.1016/j.molcel.2011.08.009
PMCID: PMC3781938  PMID: 21925381
4.  Rescue of hearing and vestibular function in a mouse model of human deafness 
Nature medicine  2013;19(3):345-350.
Hearing impairment is the most common sensory disorder, with congenital hearing impairment present in ~1 in 1000 newborns1, and yet there is no cellular cure for deafness. Hereditary deafness is often mediated by the developmental failure or degeneration of cochlear hair cells2. Until now, it was not known whether such congenital failures could be mitigated by therapeutic intervention3-5. Here we show that hearing and vestibular function can be rescued in a mouse model of human hereditary deafness. An antisense oligonucleotide (ASO) was used to correct defective pre–mRNA splicing of transcripts from the mutated USH1C.216G>A gene, which causes human Usher syndrome (Usher), the leading genetic cause of combined deafness and blindness6,7. Treatment of neonatal mice with a single systemic dose of ASO partially corrects USH1C.216G>A splicing, increases protein expression, improves stereocilia organization in the cochlea, and rescues cochlear hair cells, vestibular function and hearing in mice. Our results demonstrate the therapeutic potential of ASOs in the treatment of deafness and provide evidence that congenital deafness can be effectively overcome by treatment early in development to correct gene expression.
doi:10.1038/nm.3106
PMCID: PMC3657744  PMID: 23380860
5.  Altered Ryanodine Receptor Expression in Mild Cognitive Impairment and Alzheimer’s Disease 
Neurobiology of Aging  2011;33(5):1001.e1-1001.e6.
Intracellular Ca2+ dysregulation is an underlying component of Alzheimer’s disease (AD) pathophysiology, and recent evidence implicates the ryanodine receptor (RyR) in the disease pathway. Three genes code for different RyR isoforms and each gene transcript gives rise to several alternatively spliced mRNAs. These variants confer distinct functionality to the RyR channel, such as altering Ca2+ release properties or subcellular localization. Changes in RyR isoform expression and alternative splicing have not been examined for potential roles in AD pathogenesis. Here, we compare mRNA levels of the RyR2 and RyR3 isoforms as well as specific alternatively spliced variants across vulnerable brain regions from postmortem samples of individuals with no cognitive impairment (NCI), mild cognitive impairment (MCI) and AD. We find an increase in RyR2 transcripts in MCI brains compared to NCI. In addition, there is a reduction in a RyR2 splice variant, associated with an anti-apoptotic function, in MCI and AD brains. These alterations in RyR expression at early disease stages may reflect the onset of pathologic mechanisms leading to later neurodegeneration.
doi:10.1016/j.neurobiolaging.2011.03.011
PMCID: PMC3160507  PMID: 21531043
alternative splicing; Alzheimer’s disease; apoptosis; calcium; endoplasmic reticulum; mild cognitive impairment; pre-mRNA splicing; ryanodine receptor
6.  MicroRNAs are exported from malignant cells in customized particles 
Nucleic Acids Research  2012;40(18):9125-9138.
MicroRNAs (miRNAs) are released from cells in association with proteins or microvesicles. We previously reported that malignant transformation changes the assortment of released miRNAs by affecting whether a particular miRNA species is released or retained by the cell. How this selectivity occurs is unclear. Here we report that selectively exported miRNAs, whose release is increased in malignant cells, are packaged in structures that are different from those that carry neutrally released miRNAs (n-miRNAs), whose release is not affected by malignancy. By separating breast cancer cell microvesicles, we find that selectively released miRNAs associate with exosomes and nucleosomes. However, n-miRNAs of breast cancer cells associate with unconventional exosomes, which are larger than conventional exosomes and enriched in CD44, a protein relevant to breast cancer metastasis. Based on their large size, we call these vesicles L-exosomes. Contrary to the distribution of miRNAs among different microvesicles of breast cancer cells, normal cells release all measured miRNAs in a single type of vesicle. Our results suggest that malignant transformation alters the pathways through which specific miRNAs are exported from cells. These changes in the particles and their miRNA cargo could be used to detect the presence of malignant cells in the body.
doi:10.1093/nar/gks656
PMCID: PMC3467054  PMID: 22772984
7.  Biogenesis of mammalian microRNAs by a non-canonical processing pathway 
Nucleic Acids Research  2012;40(10):4626-4640.
Canonical microRNA biogenesis requires the Microprocessor components, Drosha and DGCR8, to generate precursor-miRNA, and Dicer to form mature miRNA. The Microprocessor is not required for processing of some miRNAs, including mirtrons, in which spliceosome-excised introns are direct Dicer substrates. In this study, we examine the processing of putative human mirtrons and demonstrate that although some are splicing-dependent, as expected, the predicted mirtrons, miR-1225 and miR-1228, are produced in the absence of splicing. Remarkably, knockout cell lines and knockdown experiments demonstrated that biogenesis of these splicing-independent mirtron-like miRNAs, termed ‘simtrons’, does not require the canonical miRNA biogenesis components, DGCR8, Dicer, Exportin-5 or Argonaute 2. However, simtron biogenesis was reduced by expression of a dominant negative form of Drosha. Simtrons are bound by Drosha and processed in vitro in a Drosha-dependent manner. Both simtrons and mirtrons function in silencing of target transcripts and are found in the RISC complex as demonstrated by their interaction with Argonaute proteins. These findings reveal a non-canonical miRNA biogenesis pathway that can produce functional regulatory RNAs.
doi:10.1093/nar/gks026
PMCID: PMC3378869  PMID: 22270084
8.  An exonic splicing enhancer within a bidirectional coding sequence regulates alternative splicing of an antisense mRNA 
RNA biology  2010;7(2):179-190.
The discovery of increasing numbers of genes with overlapping sequences highlights the problem of expression in the context of constraining regulatory elements from more than one gene. This study identifies regulatory sequences encompassed within two genes that overlap in an antisense orientation at their 3′ ends. The genes encode the α-thyroid hormone receptor gene (TRα or NR1A1) and Rev-erbα (NR1D1). In mammals TRα pre-mRNAs are alternatively spliced to yield mRNAs encoding functionally antagonistic proteins: TRα1, an authentic thyroid hormone receptor; and TRα2, a non-hormone-binding variant that acts as a repressor. TRα2-specific splicing requires two regulatory elements that overlap with Rev-erbα sequences. Functional mapping of these elements reveals minimal splicing enhancer elements that have evolved within the constraints of the overlapping Rev-erbα sequence. These results provide insight into the evolution of regulatory elements within the context of bidirectional coding sequences. They also demonstrate the ability of the genetic code to accommodate multiple layers of information within a given sequence, an important property of the code recently suggested on theoretical grounds.
PMCID: PMC2893273  PMID: 20200494
alternative splicing; exonic splicing enhancer; intronic splicing enhancer; antisense RNA; genetic code; exon evolution; nuclear receptor proteins
9.  Selective Release of MicroRNA Species from Normal and Malignant Mammary Epithelial Cells 
PLoS ONE  2010;5(10):e13515.
MicroRNAs (miRNAs) in body fluids are candidate diagnostics for a variety of conditions and diseases, including breast cancer. One premise for using extracellular miRNAs to diagnose disease is the notion that the abundance of the miRNAs in body fluids reflects their abundance in the abnormal cells causing the disease. As a result, the search for such diagnostics in body fluids has focused on miRNAs that are abundant in the cells of origin. Here we report that released miRNAs do not necessarily reflect the abundance of miRNA in the cell of origin. We find that release of miRNAs from cells into blood, milk and ductal fluids is selective and that the selection of released miRNAs may correlate with malignancy. In particular, the bulk of miR-451 and miR-1246 produced by malignant mammary epithelial cells was released, but the majority of these miRNAs produced by non-malignant mammary epithelial cells was retained. Our findings suggest the existence of a cellular selection mechanism for miRNA release and indicate that the extracellular and cellular miRNA profiles differ. This selective release of miRNAs is an important consideration for the identification of circulating miRNAs as biomarkers of disease.
doi:10.1371/journal.pone.0013515
PMCID: PMC2958125  PMID: 20976003
10.  A feedback loop regulates splicing of the spinal muscular atrophy-modifying gene, SMN2 
Human Molecular Genetics  2010;19(24):4906-4917.
Spinal muscular atrophy (SMA) is a neurological disorder characterized by motor neuron degeneration and progressive muscle paralysis. The disease is caused by a reduction in survival of motor neuron (SMN) protein resulting from homozygous deletion of the SMN1 gene. SMN protein is also encoded by SMN2. However, splicing of SMN2 exon 7 is defective, and consequently, the majority of the transcripts produce a truncated, unstable protein. SMN protein itself has a role in splicing. The protein is required for the biogenesis of spliceosomal snRNPs, which are essential components of the splicing reaction. We now show that SMN protein abundance affects the splicing of SMN2 exon 7, revealing a feedback loop inSMN expression. The reduced SMN protein concentration observed in SMA samples and in cells depleted of SMN correlates with a decrease in cellular snRNA levels and a decrease in SMN2 exon 7 splicing. Furthermore, altering the relative abundance or activity of individual snRNPs has distinct effects on exon 7 splicing, demonstrating that core spliceosomal snRNPs influence SMN2 alternative splicing. Our results identify a feedback loop in SMN expression by which low SMN protein levels exacerbate SMN exon 7 skipping, leading to a further reduction in SMN protein. These results imply that a modest increase in SMN protein abundance may cause a disproportionately large increase in SMN expression, a finding that is important for assessing the therapeutic potential of SMA treatments and understanding disease pathogenesis.
doi:10.1093/hmg/ddq425
PMCID: PMC2989896  PMID: 20884664
11.  Tetracyclines That Promote SMN2 Exon 7 Splicing as Therapeutics for Spinal Muscular Atrophy 
There is at present no cure or effective therapy for spinal muscular atrophy (SMA), a neurodegenerative disease that is the leading genetic cause of infant mortality. SMA usually results from loss of the SMN1 (survival of motor neuron 1) gene, which leads to selective motor neuron degeneration. SMN2 is nearly identical to SMN1 but has a nucleotide replacement that causes exon 7 skipping, resulting in a truncated, unstable version of the SMA protein. SMN2 is present in all SMA patients, and correcting SMN2 splicing is a promising approach for SMA therapy. We identified a tetracycline-like compound, PTK-SMA1, which stimulates exon 7 splicing and increases SMN protein levels in vitro and in vivo in mice. Unlike previously identified molecules that stimulate SMN production via SMN2 promoter activation or undefined mechanisms, PTK-SMA1 is a unique therapeutic candidate in that it acts by directly stimulating splicing of exon 7. Synthetic small-molecule compounds such as PTK-SMA1 offer an alternative to antisense oligonucleotide therapies that are being developed as therapeutics for a number of disease-associated splicing defects.
doi:10.1126/scitranslmed.3000208
PMCID: PMC2818805  PMID: 20161659
12.  Control of Pre-mRNA Splicing by the General Splicing Factors PUF60 and U2AF65 
PLoS ONE  2007;2(6):e538.
Pre-mRNA splicing is a crucial step in gene expression, and accurate recognition of splice sites is an essential part of this process. Splice sites with weak matches to the consensus sequences are common, though it is not clear how such sites are efficiently utilized. Using an in vitro splicing-complementation approach, we identified PUF60 as a factor that promotes splicing of an intron with a weak 3′ splice-site. PUF60 has homology to U2AF65, a general splicing factor that facilitates 3′ splice-site recognition at the early stages of spliceosome assembly. We demonstrate that PUF60 can functionally substitute for U2AF65 in vitro, but splicing is strongly stimulated by the presence of both proteins. Reduction of either PUF60 or U2AF65 in cells alters the splicing pattern of endogenous transcripts, consistent with the idea that regulation of PUF60 and U2AF65 levels can dictate alternative splicing patterns. Our results indicate that recognition of 3′ splice sites involves different U2AF-like molecules, and that modulation of these general splicing factors can have profound effects on splicing.
doi:10.1371/journal.pone.0000538
PMCID: PMC1888729  PMID: 17579712
13.  Comprehensive splice-site analysis using comparative genomics 
Nucleic Acids Research  2006;34(14):3955-3967.
We have collected over half a million splice sites from five species—Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans and Arabidopsis thaliana—and classified them into four subtypes: U2-type GT–AG and GC–AG and U12-type GT–AG and AT–AC. We have also found new examples of rare splice-site categories, such as U12-type introns without canonical borders, and U2-dependent AT–AC introns. The splice-site sequences and several tools to explore them are available on a public website (SpliceRack). For the U12-type introns, we find several features conserved across species, as well as a clustering of these introns on genes. Using the information content of the splice-site motifs, and the phylogenetic distance between them, we identify: (i) a higher degree of conservation in the exonic portion of the U2-type splice sites in more complex organisms; (ii) conservation of exonic nucleotides for U12-type splice sites; (iii) divergent evolution of C.elegans 3′ splice sites (3′ss) and (iv) distinct evolutionary histories of 5′ and 3′ss. Our study proves that the identification of broad patterns in naturally-occurring splice sites, through the analysis of genomic datasets, provides mechanistic and evolutionary insights into pre-mRNA splicing.
doi:10.1093/nar/gkl556
PMCID: PMC1557818  PMID: 16914448

Results 1-13 (13)