pri-miRNA Transcripts Research Articles

Functional Dissection of pri-miR-290~295 in Dgcr8 Knockout Mouse Embryonic Stem Cells.

The DiGeorge syndrome critical region gene 8 (Dgcr8) knockout strategy has been widely used to study the function of canonical microRNAs (miRNAs) in vitro and in vivo. However, primary miRNA (pri-miRNA) transcripts are accumulated in Dgcr8 knockout cells due to interrupted processing. Whether abnormally accumulated pri-miRNAs have any function is unknown. Here, using clustered regularly interspaced short palindromic repeats system/CRISPR-associated protein 9 (CRISPR/Cas9), we successfully knocked out the primary microRNA-290~295 (pri-miR-290~295) cluster, the most highly expressed miRNA cluster in mouse embryonic stem cells (ESCs), in Dgcr8 knockout background. We found that the major defects associated with Dgcr8 knockout in mouse ESCs, including higher expression of epithelial-to-mesenchymal transition (EMT) markers, slower proliferation, G1 accumulation, and defects in silencing self-renewal, were not affected by the deletion of pri-miR-290~290 cluster. Interestingly, the transcription of neighboring gene nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 12(Nlrp12) was upregulated upon the deletion of the pri-miR-290~295 cluster. Together, our results suggested that the major defects in Dgcr8 knockout ESCs were not due to the accumulation of pri-miR-290~295, and the deletion of miRNA genes could affect the transcription of neighboring DNA elements.

Put the Pedal to the METTL1: Adding Internal m7G Increases mRNA Translation Efficiency and Augments miRNA Processing.

Complementary papers by Zhang, Liu, and colleagues (Zhang etal., 2019) and Pandolfini, Barbieri, and colleagues (Pandolfini etal., 2019) develop new sequencing techniques that reveal that METTL1 N7-methylates internal guanosines in mRNAs and miRNAs to increase translation efficiency and miRNA processing, respectively.

Drosophila Trf4-1 involves in mRNA and primary miRNA transcription

Drosophila Trf4-1 (DmTrf4-1) is a polyadenylation polymerase or terminal nucleotidyl transferase (PAP/TENT) that has been reported to add poly(A) tails to snRNAs in nucleus or mRNAs in cytoplasm. Here, we found that the loss of Trf4-1 resulted in the reduction of mRNAs and primary miRNAs (pri-miRNAs) in both Drosophila S2 cells and adult flies. Interestingly, the role of Trf4-1 in transcription is independent of its PAP/TENT activity. Moreover, using the chromatin immunoprecipitation assay, we uncovered that the loss of Trf4-1 led to abnormal RNA polymerase II accumulation and reduced H3K4me3 binding in promoter regions. Thus, our study indicates a positive role of Trf4-1 in the transcription of mRNAs and pri-miRNAs.

Bioinformatics Analysis of the Novel Conserved Micropeptides Encoded by the Plants of Family Brassicaceae

Background: The new class of plant small peptide regulators was recently shown to be encoded by primiRNA transcripts which can be transported to cytoplasm in unprocessed mRNA-like form. Striking similarities in general phenotypic activities between human miR200a/b and plant miR156a suggested us that a comparison with the coding potential between the corresponding pri-miRNAs could identify parallels in the encoded miPEPs. Method: The study aimed to explore the protein coding ability of the pri-miR156a in Brassicaceae plants using bioinformatics analysis of the available proteomes and translatomes reported for Arabidopsis thaliana. Also, the physicochemical parameters of miPEP-156a were examined. Results: Our analysis showed that predicted miPEP-156a micropeptide is evolutionarily conserved in plant family Brassicaceae. We propose that functional properties of miPEP-156a can be affected by posttranslational modifications. Conclusion: Despite the well-known fact that primiRNAs are acting as non-protein-coding RNAs, the published data suggest that, in the plant genomes, some pri-miRNAs can also be found in polysomes, and the expression of these miRNA precursors may results in formation of micropeptides which may be involved in regulation of gene expression.

Efficient Knockdown and Lack of Passenger Strand Activity by Dicer-Independent shRNAs Expressed from Pol II-Driven MicroRNA Scaffolds

The expression of short hairpin RNAs (shRNAs) may result in unwanted activity from the co-processed passenger strand. Recent studies have shown that shortening the stem of conventional shRNAs abolishes passenger strand release. These Dicer-independent shRNAs, expressed from RNA polymerase III (Pol III) promoters, rely on Ago2 processing in resemblance to miR-451. Using strand-specific reporters, we tested two designs, and our results support the loss of passenger strand activity. We demonstrate that artificial primary microRNA (pri-miRNA) transcripts, expressed from Pol II promoters, can potently silence a gene of choice. Among six different scaffolds tested, miR-324 and miR-451 were readily re-targeted to direct efficient knockdown from either a CMV or a U1 snRNA promoter. Importantly, the miR-shRNAs have no passenger strand activity and remain active in Dicer-knockout cells. Our vectors are straightforward to design, as we replace the pre-miR-324 or -451 sequences with a Dicer-independent shRNA mimicking miR-451 with unpaired A-C nucleotides at the base. The use of Pol II promoters allows for controlled expression, while the inclusion of pri-miRNA sequences likely requires Drosha processing and, as such, mimics microRNA biogenesis. Since this improved and tunable system bypasses the requirement for Dicer activity and abolishes passenger strand activity completely, it will likely prove favorable in both research and therapeutic applications in terms of versatility and enhanced safety.

MAC3A and MAC3B, Two Core Subunits of the MOS4-Associated Complex, Positively Influence miRNA Biogenesis.

MAC3A and MAC3B are conserved U-box-containing proteins in eukaryotes. They are subunits of the MOS4-associated complex (MAC) that plays essential roles in plant immunity and development in Arabidopsis thaliana However, their functional mechanisms remain elusive. Here, we show that Arabidopsis MAC3A and MAC3B act redundantly in microRNA (miRNA) biogenesis. Lack of both MAC3A and MAC3B in the mac3b mac3b double mutant reduces the accumulation of miRNAs, causing elevated transcript levels of miRNA targets. mac3a mac3b also decreases the levels of primary miRNA transcripts (pri-miRNAs). However, MAC3A and MAC3B do not affect the promoter activity of genes encoding miRNAs (MIR genes), suggesting that they may not affect MIR transcription. This result, together with the fact that MAC3A associates with pri-miRNAs in vivo, indicates that MAC3A and MAC3B may stabilize pri-miRNAs. Furthermore, we find that MAC3A and MAC3B interact with the DCL1 complex that catalyzes miRNA maturation, promote DCL1 activity, and are required for the localization of HYL1, a component of the DCL1 complex. Besides MAC3A and MAC3B, two other MAC subunits, CDC5 and PRL1, also function in miRNA biogenesis. Based on these results, we propose that MAC functions as a complex to control miRNA levels through modulating pri-miRNA transcription, processing, and stability.

Identifying Pri-miRNA Transcription Start Sites.

MicroRNAs (miRNAs) are small non-coding RNAs that can regulate gene expression playing vital role in nearly all biological pathways. Even though miRNAs have been intensely studied for more than two decades, information regarding miRNA transcription regulation remains limited. The rapid cleavage of primary miRNA transcripts (pri-miRNAs) by Drosha in the nucleus hinders their identification with conventional RNA-seq approaches. Identifying the transcription start site (TSS) of miRNAs will enable genome-wide identification of their expression regulators, including transcription factors (TFs), other non-coding RNAs (ncRNAs) and epigenetic modifiers, providing significant breakthroughs in understanding the mechanisms underlying miRNA expression in development and disease. Here we present a protocol that utilizes microTSS, a versatile computational framework for accurate and single-nucleotide resolution miRNA TSS predictions as well as miRGen, a database of miRNA gene TSSs coupled with genome-wide maps of TF binding sites.

Analysis of primary microRNA loci from nascent transcriptomes reveals regulatory domains governed by chromatin architecture.

Changes in mature microRNA (miRNA) levels that occur downstream of signaling cascades play an important role during human development and disease. However, the regulation of primary microRNA (pri-miRNA) genes remains to be dissected in detail. To address this, we followed a data-driven approach and developed a transcript identification, validation and quantification pipeline for characterizing the regulatory domains of pri-miRNAs. Integration of 92 nascent transcriptomes and multilevel data from cells arising from ecto-, endo- and mesoderm lineages reveals cell type-specific expression patterns, allows fine-resolution mapping of transcription start sites (TSS) and identification of candidate regulatory regions. We show that inter- and intragenic pri-miRNA transcripts span vast genomic regions and active TSS locations differ across cell types, exemplified by the mir-29a∼29b-1, mir-100∼let-7a-2∼125b-1 and miR-221∼222 clusters. Considering the presence of multiple TSS as an important regulatory feature at miRNA loci, we developed a strategy to quantify differential TSS usage. We demonstrate that the TSS activities associate with cell type-specific super-enhancers, differential stimulus responsiveness and higher-order chromatin structure. These results pave the way for building detailed regulatory maps of miRNA loci.

The Arabidopsis MOS4-Associated Complex Promotes MicroRNA Biogenesis and Precursor Messenger RNA Splicing.

In Arabidopsis thaliana, the MOS4-ASSOCIATED COMPLEX (MAC) is required for defense and development. The evolutionarily conserved, putative RNA helicase MAC7 is a component of the Arabidopsis MAC, and the human MAC7 homolog, Aquarius, is implicated in pre-mRNA splicing. Here, we show that mac7-1, a partial loss-of-function mutant in MAC7, and two other MAC subunit mutants, mac3a mac3b and prl1 prl2 (pleiotropic regulatory locus), exhibit reduced microRNA (miRNA) levels, indicating that MAC promotes miRNA biogenesis. The mac7-1 mutant shows reduced primary miRNA (pri-miRNA) levels without affecting miRNA gene (MIR) promoter activity or the half-life of pri-miRNA transcripts. As a nuclear protein, MAC7 is not concentrated in dicing bodies, but it affects the localization of HYPONASTIC LEAVES1 (HYL1), a key protein in pri-miRNA processing, to dicing bodies. Immunoprecipitation of HYL1 retrieved 11 known MAC subunits, including MAC7, indicating association between HYL1 and MAC. We propose that MAC7 links MIR transcription to pri-miRNA processing. RNA-seq analysis showed that downregulated genes in MAC subunit mutants are mostly involved in plant defense and stimulus responses, confirming a role of MAC in biotic and abiotic stress responses. We also discovered global intron retention defects in mutants in three subunits of MAC, thus linking MAC function to splicing in Arabidopsis.

Analysis of primary microRNA loci from nascent transcriptomes reveals regulatory domains governed by chromatin architecture.

Changes in mature microRNA (miRNA) levels that occur downstream of signaling cascades play an important role during human development and disease. However, the regulation of primary microRNA (pri-miRNA) genes remains to be dissected in detail. To address this, we followed a data-driven approach and developed a transcript identification, validation and quantification pipeline for characterizing the regulatory domains of pri-miRNAs. Integration of 92 nascent transcriptomes and multilevel data from cells arising from ecto-, endo- and mesoderm lineages reveals cell type-specific expression patterns, allows fine-resolution mapping of transcription start sites (TSS) and identification of candidate regulatory regions. We show that inter- and intragenic pri-miRNA transcripts span vast genomic regions and active TSS locations differ across cell types, exemplified by the mir-29a∼29b-1, mir-100∼let-7a-2∼125b-1 and miR-221∼222 clusters. Considering the presence of multiple TSS as an important regulatory feature at miRNA loci, we developed a strategy to quantify differential TSS usage. We demonstrate that the TSS activities associate with cell type-specific super-enhancers, differential stimulus responsiveness and higher-order chromatin structure. These results pave the way for building detailed regulatory maps of miRNA loci.

The Emerging Role of miR-200 Family in Cardiovascular Diseases.

Recent studies have shown that reactive oxygen species increase the expression of miR-200 family (miR-200); however, little is known about this micro-RNA family in the cardiovascular system. In this Viewpoint, we provide evidence suggesting that miR-200 may be important in conditions that affect the heart and blood vessels. Oxidative stress plays a major role in cardiovascular physiopathology and some common conditions including aging, diabetes mellitus, atherosclerosis, and reperfusion injury induce reactive oxygen species (ROS). In 2011, it was first published that endothelial and skeletal muscle cells exposed to oxidative stress, in vitro and in vivo, exhibit a marked increase in miR-2001. Subsequently, ROS ability to induce miR-200 has been confirmed in many cell types. Furthermore, hypoxia enhances miR-429, an miR-200 member, and miR-429 targets HIF-1α (hypoxia-inducible factor-1α) and decreases its expression. Most miR-200 studies have been in the cancer field: miR-200 modulates epithelial–mesenchymal transition by targeting the transcription factors ZEB1 and ZEB2 (Zinc-finger E-box binding homeobox 1 and 2) and circulating miR-200 may represent clinically useful cancer biomarkers. In contrast, the role of miR-200 in CV diseases is still poorly investigated. MiRNAs are short (21–22 nucleotides) noncoding RNAs: their seed sequence (nucleotides 2–8 from the 5′ end) targets specific mRNAs and via this mechanism they inhibit translation and also may modulate mRNA stability. The miR-200 family is composed of 5 members clustered and expressed as 2 separate polycistronic pri-miRNA transcripts: in humans, miR-200c and miR-141 are on chromosome 12; miR-200a, miR-200b, and miR-429 are on chromosome 1. They are also identified by different seed sequences: subgroup I comprises miR-141 and miR-200a; subgroup II includes miR-200b, miR-200c, and miR-429. Because each miRNA can target numerous mRNAs and the 2 miR-200 subgroups have different seed sequences, the functional impact of miR-200 can be profound. The Figure shows the major miR-200c–activated …

STV1, a ribosomal protein, binds primary microRNA transcripts to promote their interaction with the processing complex in Arabidopsis

MicroRNAs (miRNAs) are key regulators of gene expression. They are processed from primary miRNA transcripts (pri-miRNAs), most of which are transcribed by DNA-dependent polymerase II (Pol II). miRNA levels are precisely controlled to maintain various biological processes. Here, we report that SHORT VALVE 1 (STV1), a conserved ribosomal protein, acts in miRNA biogenesis in Arabidopsis A portion of STV1 localizes in the nucleus and binds pri-miRNAs. Using pri-miR172b as a reporter, we show that STV1 binds the stem-loop flanked by a short 5' arm within pri-miRNAs. Lack of STV1 reduces the association of pri-miRNAs with their processing complex. These data suggest that STV1 promotes miRNA biogenesis through facilitating the recruitment of pri-miRNAs to their processing complex. Furthermore, we show that STV1 indirectly involves in the occupancy of Pol II at the promoters of miRNA coding genes (MIR) and influences MIR promoter activities. Based on these results, we propose that STV1 refines the accumulation of miRNAs through its combined effects on pri-miRNA processing and transcription. This study uncovers an extraribosomal function of STV1.

RNA-binding protein DUS16 plays an essential role in primary miRNA processing in the unicellular alga Chlamydomonas reinhardtii

Canonical microRNAs (miRNAs) are embedded in duplexed stem-loops in long precursor transcripts and are excised by sequential cleavage by DICER nuclease(s). In this miRNA biogenesis pathway, dsRNA-binding proteins play important roles in animals and plants by assisting DICER. However, these RNA-binding proteins are poorly characterized in unicellular organisms. Here we report that a unique RNA-binding protein, Dull slicer-16 (DUS16), plays an essential role in processing of primary-miRNA (pri-miRNA) transcripts in the unicellular green alga Chlamydomonas reinhardtii In animals and plants, dsRNA-binding proteins involved in miRNA biogenesis harbor two or three dsRNA-binding domains (dsRBDs), whereas DUS16 contains one dsRBD and also an ssRNA-binding domain (RRM). The null mutant of DUS16 showed a drastic reduction in most miRNA species. Production of these miRNAs was complemented by expression of full-length DUS16, but the expression of RRM- or dsRBD-truncated DUS16 did not restore miRNA production. Furthermore, DUS16 is predominantly localized to the nucleus and associated with nascent (unspliced form) pri-miRNAs and the DICER-LIKE 3 protein. These results suggest that DUS16 recognizes pri-miRNA transcripts cotranscriptionally and promotes their processing into mature miRNAs as a component of a microprocessor complex. We propose that DUS16 is an essential factor for miRNA production in Chlamydomonas and, because DUS16 is functionally similar to the dsRNA-binding proteins involved in miRNA biogenesis in animals and land plants, our report provides insight into this mechanism in unicellular eukaryotes.

HP1BP3, a Chromatin Retention Factor for Co-transcriptional MicroRNA Processing

Recent studies suggest that the microprocessor (Drosha-DGCR8) complex can be recruited to chromatin to catalyze co-transcriptional processing ofprimary microRNAs (pri-miRNAs) in mammalian cells. However, the molecular mechanism of co-transcriptional miRNA processing is poorly understood. Here we find that HP1BP3, a histone H1-like chromatin protein, specifically associates with the microprocessor and promotes global miRNA biogenesis inhuman cells. Chromatin immunoprecipitation (ChIP) studies reveal genome-wide co-localization of HP1BP3 and Drosha and HP1BP3-dependent Drosha binding to actively transcribed miRNA loci. Moreover, HP1BP3 specifically binds endogenous pri-miRNAs and facilitates the Drosha/pri-miRNA association invivo. Knockdown of HP1BP3 compromises pri-miRNA processing by causing premature release of pri-miRNAs from the chromatin. Taken together, these studies suggest that HP1BP3 promotes co-transcriptional miRNA processing via chromatin retention of nascent pri-miRNA transcripts. This work significantly expands the functional repertoire of the H1 family of proteins and suggests the existence of chromatin retention factors for widespread co-transcriptional miRNA processing.

MiR-125b-1 is repressed by histone modifications in breast cancer cell lines.

PurposeDownregulation of miR-125b-1 is associated with poor prognosis in breast cancer patients. In this work we investigated the effect of histone modifications on the regulation of this gene promoter.Methods and resultsWe evaluated the enrichment of two histone modifications involved in gene repression, H3K9me3 and H3K27me3, on the miR-125b-1 promoter in two breast cancer cell lines, MCF7 (luminal A subtype) and MDA-MB-231 (triple-negative subtype), compared to the non-transformed breast cell line MCF10A. H3K27me3 and H3K9me3 were enriched in MCF7 and MDA-MB-231 cells, respectively. Next, we used an EZH2 inhibitor to examine the reactivation of miR-125b-1 in MCF7 cells and evaluated the transcriptional levels of pri-miR-125b-1 and mature miR-125b by qRT-PCR. pri-miRNA and mature miRNA transcripts were both increased after treatment of MCF7 cells with the EZH2 inhibitor, whereas no effect on miR-125b-1 expression levels was observed in MDA-MB-231 and MCF10A cells. We subsequently evaluated the effect of miR-125b-1 reactivation on the expression and protein levels of BAK1, a target of miR-125b. We observed 60 and 70 % decreases in the expression and protein levels of BAK1, respectively, compared to cells that were not treated with the EZH2 inhibitor. We over-expressed KDM4B/JMJD2B to reactivate this miRNA, resulting in a three-fold increase in miR-125b expression compared with the same cell line without KDM4B/JMJD2B over-expression.ConclusionThe miR-125b-1 is repressed by different epigenetic mechanisms depending on the breast cancer subtype and that miR-125b-1 reactivation specifically eliminates the effect of repressive histone modifications on the expression of an pro-apoptotic target.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-2475-z) contains supplementary material, which is available to authorized users.

MicroRNA Expression during Bovine Oocyte Maturation and Fertilization.

Successful fertilization and subsequent embryo development rely on complex molecular processes starting with the development of oocyte competence through maturation. MicroRNAs (miRNAs) are small non-coding RNA molecules that function as gene regulators in many biological systems, including the oocyte and embryo. In order to further explore the roles of miRNAs in oocyte maturation, we employed small RNA sequencing as a screening tool to identify and characterize miRNA populations present in pools of bovine germinal vesicle (GV) oocytes, metaphase II (MII) oocytes, and presumptive zygotes (PZ). Each stage contained a defined miRNA population, some of which showed stable expression while others showed progressive changes between stages that were subsequently confirmed by quantitative reverse transcription polymerase chain reaction (RT-PCR). Bta-miR-155, bta-miR-222, bta-miR-21, bta-let-7d, bta-let-7i, and bta-miR-190a were among the statistically significant differentially expressed miRNAs (p < 0.05). To determine whether changes in specific primary miRNA (pri-miRNA) transcripts were responsible for the observed miRNA changes, we evaluated pri-miR-155, -222 and let-7d expression. Pri-miR-155 and -222 were not detected in GV oocytes but pri-miR-155 was present in MII oocytes, indicating transcription during maturation. In contrast, levels of pri-let-7d decreased during maturation, suggesting that the observed increase in let-7d expression was likely due to processing of the primary transcript. This study demonstrates that both dynamic and stable populations of miRNAs are present in bovine oocytes and zygotes and extend previous studies supporting the importance of the small RNA landscape in the maturing bovine oocyte and early embryo.

Small RNA sequencing of colorectal cancer to reveal differences in microRNA expression between normal and tumor tissue.

506 Background: MicroRNAs (miRNAs) are promising biomarkers and therapeutic agents for colorectal cancer (CRC). However, much remains to be known about the biology of miRNAs and miRNA variants in tumor tissue and why the expression differs from normal tissue. Methods: RNA from 48 normal and 48 cancer tissue samples were sequenced using the Illumina small RNA sequencing protocol. Samples were taken from both male and female patients with tumor stage 1-4. Results: We show that almost 50% of microRNAs (miRNAs) are differentially expressed between normal and cancer tissue. When analyzing pre-miRNAs that map to genomic clusters, so-called clustered miRNAs, we show that miRNAs in the same cluster often have similar expression profiles. This shows that common changes in co-expressed miRNAs seem to explain some of the miRNA expression changes between normal and tumor samples and indicates that altered pri-miRNA transcription is the mechanism for at least some of the miRNA changes. Analyses of miRNA variants, so-called isomiRs, revealed large variations between tumor and normal samples, similar to those observed for the canonical miRNAs. Different isomiR types show different expression profiles in tumor vs normal samples, indicating that miRNA processing is altered in tumor cells. Finally, we show that several canonical miRNAs and isomiRs correlate with genetic signatures in the tumor samples, including KRAS and BRAF mutation, Microsatellite instability and MLH1 methylation, indicating that tumor subtypes have different miRNA expression. Conclusions: Our results show that miRNA expression predicts tumor subtypes with different genetic signatures. We show that some of the changes in miRNA expression are due to different transcription programs in cancer vs normal tissue.

Transcriptional, post-transcriptional and chromatin-associated regulation of pri-miRNAs, pre-miRNAs and moRNAs.

MicroRNAs (miRNAs) play a major role in the post-transcriptional regulation of target genes, especially in development and differentiation. Our understanding about the transcriptional regulation of miRNA genes is limited by inadequate annotation of primary miRNA (pri-miRNA) transcripts. Here, we used CAGE-seq and RNA-seq to provide genome-wide identification of the pri-miRNA core promoter repertoire and its dynamic usage during zebrafish embryogenesis. We assigned pri-miRNA promoters to 152 precursor-miRNAs (pre-miRNAs), the majority of which were supported by promoter associated post-translational histone modifications (H3K4me3, H2A.Z) and RNA polymerase II (RNAPII) occupancy. We validated seven miR-9 pri-miRNAs by in situ hybridization and showed similar expression patterns as mature miR-9. In addition, processing of an alternative intronic promoter of miR-9–5 was validated by 5′ RACE PCR. Developmental profiling revealed a subset of pri-miRNAs that are maternally inherited. Moreover, we show that promoter-associated H3K4me3, H2A.Z and RNAPII marks are not only present at pri-miRNA promoters but are also specifically enriched at pre-miRNAs, suggesting chromatin level regulation of pre-miRNAs. Furthermore, we demonstrated that CAGE-seq also detects 3′-end processing of pre-miRNAs on Drosha cleavage site that correlates with miRNA-offset RNAs (moRNAs) production and provides a new tool for detecting Drosha processing events and predicting pre-miRNA processing by a genome-wide assay.

Genome-wide annotation of microRNA primary transcript structures reveals novel regulatory mechanisms.

Precise regulation of microRNA (miRNA) expression is critical for diverse physiologic and pathophysiologic processes. Nevertheless, elucidation of the mechanisms through which miRNA expression is regulated has been greatly hindered by the incomplete annotation of primary miRNA (pri-miRNA) transcripts. While a subset of miRNAs are hosted in protein-coding genes, the majority of pri-miRNAs are transcribed as poorly characterized noncoding RNAs that are 10's to 100's of kilobases in length and low in abundance due to efficient processing by the endoribonuclease DROSHA, which initiates miRNA biogenesis. Accordingly, these transcripts are poorly represented in existing RNA-seq data sets and exhibit limited and inaccurate annotation in current transcriptome assemblies. To overcome these challenges, we developed an experimental and computational approach that allows genome-wide detection and mapping of pri-miRNA structures. Deep RNA-seq in cells expressing dominant-negative DROSHA resulted in much greater coverage of pri-miRNA transcripts compared with standard RNA-seq. A computational pipeline was developed that produces highly accurate pri-miRNA assemblies, as confirmed by extensive validation. This approach was applied to a panel of human and mouse cell lines, providing pri-miRNA transcript structures for 1291/1871 human and 888/1181 mouse miRNAs, including 594 human and 425 mouse miRNAs that fall outside protein-coding genes. These new assemblies uncovered unanticipated features and new potential regulatory mechanisms, including links between pri-miRNAs and distant protein-coding genes, alternative pri-miRNA splicing, and transcripts carrying subsets of miRNAs encoded by polycistronic clusters. These results dramatically expand our understanding of the organization of miRNA-encoding genes and provide a valuable resource for the study of mammalian miRNA regulation.

The RNA-binding protein HOS5 and serine/arginine-rich proteins RS40 and RS41 participate in miRNA biogenesis in Arabidopsis.

MicroRNAs are a class of small regulatory RNAs that are generated from primary miRNA (pri-miRNA) transcripts with a stem-loop structure. Accuracy of the processing of pri-miRNA into mature miRNA in plants can be enhanced by SERRATE (SE) and HYPONASTIC LEAVES 1 (HYL1). HYL1 activity is regulated by the FIERY2 (FRY2)/RNA polymerase II C-terminal domain phosphatase-like 1 (CPL1). Here, we discover that HIGH OSMOTIC STRESS GENE EXPRESSION 5 (HOS5) and two serine/arginine-rich splicing factors RS40 and RS41, previously shown to be involved in pre-mRNA splicing, affect the biogenesis of a subset of miRNA. These proteins are required for correct miRNA strand selection and the maintenance of miRNA levels. FRY2 dephosphorylates HOS5 whose phosphorylation status affects its subnuclear localization. HOS5 and the RS proteins bind both intronless and intron-containing pri-miRNAs. Importantly, all of these splicing-related factors directly interact with both HYL1 and SE in nuclear splicing speckles. Our results indicate that these splicing factors are directly involved in the biogenesis of a group of miRNA.