Drosha Cleavage Site Research Articles

Extended haplotype with rs41524547-G defines the ancestral origin of SCA10.

Spinocerebellar ataxia type 10 (SCA10) is a rare autosomal dominant ataxia caused by a large expansion of the (ATTCT)n repeat in ATXN10. SCA10 was described in Native American and Asian individuals which prompted a search for an expanded haplotype to confirm a common ancestral origin for the expansion event. All patients with SCA10 expansions in our cohort share a single haplotype defined at the 5'-end by the minor allele of rs41524547, located ~35kb upstream of the SCA10 expansion. Intriguingly, rs41524547 is located within the miRNA gene, MIR4762, within its DROSHA cleavage site and just outside the seed sequence for mir4792-5p. The world-wide frequency of rs41524547-G is less than 5% and found almost exclusively in the Americas and East Asia-a geographic distribution that mirrors reported SCA10 cases. We identified rs41524547-G(+) DNA from the 1000 Genomes/International Genome Sample Resource and our own general population samples and identified SCA10 repeat expansions in up to 25% of these samples. The reduced penetrance of these SCA10 expansions may be explained by a young (pre-onset) age at sample collection, a small repeat size, purity of repeat units, or the disruption of miR4762-5p function. We conclude that rs41524547-G is the most robust at-risk SNP allele for SCA10, is useful for screening of SCA10 expansions in population genetics studies and provides the most compelling evidence to date for a single, prehistoric origin of SCA10 expansions sometime prior to or during the migration of individuals across the Bering Land Bridge into the Americas.

SRSF3 shapes the structure of miR‐17‐92 cluster RNA and promotes selective processing of miR‐17 and miR‐20a

MicroRNA (miRNA) biogenesis is tightly regulated to maintain distinct miRNA expression patterns. Almost half of mammalian miRNAs are generated from miRNA clusters, but this process is not well understood. We show here that Serine‐arginine rich splicing factor 3 (SRSF3) controls the processing of miR‐17‐92 cluster miRNAs in pluripotent and cancer cells. SRSF3 binding to multiple CNNC motifs downstream of Drosha cleavage sites within miR‐17‐92 is required for the efficient processing of the cluster. SRSF3 depletion specifically compromises the processing of two paralog miRNAs, miR‐17 and miR‐20a. In addition to SRSF3 binding to the CNNC sites, the SRSF3 RS‐domain is essential for miR‐17‐92 processing. SHAPE‐MaP probing demonstrates that SRSF3 binding disrupts local and distant base pairing, resulting in global changes in miR‐17‐92 RNA structure. Our data suggest a model where SRSF3 binding, and potentially its RS‐domain interactions, may facilitate an RNA structure that promotes miR‐17‐92 processing. SRSF3‐mediated increase in miR‐17/20a levels inhibits the cell cycle inhibitor p21, promoting self‐renewal in normal and cancer cells. The SRSF3‐miR‐17‐92‐p21 pathway operates in colorectal cancer, linking SRSF3‐mediated pri‐miRNA processing and cancer pathogenesis.

The context signals of mitochondrial miRNAs (mitomiRs) of mammals.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level in the cytoplasm and play an important role in a wide range of biological processes. Recent studies have found that the miRNA sequences are presented not only in the cytoplasm, but also in the mitochondria. These miRNAs (the so-called mitomiRs) may be the sequences of nuclear or mitochondrial origin; some of them are involved in regulation of the mitochondrial gene functions, while the role of others is still unknown. The identification of nucleotide signals, which are unique to mitomiRs, may help to determine this role. We formed a dataset that combined the experimentally discovered mitomiRs in human, rat and mouse. To isolate signals that may be responsible for the mitomiRs' functions or for their translocation from or into mitochondria a context analysis was carried out for the sequences. For three species in the group mitomiRs/non-mitomiRs and the group of all miRNAs from the miRBase database statistically overrepresented 8-letter motifs were identified (p-value < 0.01 with Bonferroni correction for multiple comparisons), for these motifs the patterns of the localization in functionally important regions for different types of miRNAs were found. Also, for the group mitomiRs/non-mitomiRs we found the statistically significant features of the miRNA nucleotide context near the Dicer and Drosha cleavage sites (Pearson's χ2 test of independence for the first three positions of the miRNA, p-value < 0.05). The observed nucleotide frequencies may indicate a more homogeneous pri-miRNA cleavage by the Drosha complex during the formation of the 5' end of mitomiRs. The obtained results can help to determine the role of the nucleotide signals in the origin, processing, and functions of the mitomiRs.

Solution Structure of NPSL2, A Regulatory Element in the oncomiR-1 RNA

The miR-17 ∼ 92a polycistron, also known as oncomiR-1, is commonly overexpressed in multiple cancers and has several oncogenic properties. OncomiR-1 encodes six constituent microRNAs (miRs), each enzymatically processed with different efficiencies. However, the structural mechanism that regulates this differential processing remains unclear. Chemical probing of oncomiR-1 revealed that the Drosha cleavage sites of pri-miR-92a are sequestered in a four-way junction. NPSL2, an independent stem loop element, is positioned just upstream of pri-miR-92a and sequesters a crucial part of the sequence that constitutes the basal helix of pri-miR-92a. Disruption of the NPSL2 hairpin structure could promote the formation of a pri-miR-92a structure that is primed for processing by Drosha. Thus, NPSL2 is predicted to function as a structural switch, regulating pri-miR-92a processing. Here, we determined the solution structure of NPSL2 using solution NMR spectroscopy. This is the first high-resolution structure of an oncomiR-1 element. NPSL2 adopts a hairpin structure with a large, but highly structured, apical and internal loops. The 10-bp apical loop contains a pH-sensitive A+·C mismatch. Additionally, several adenosines within the apical and internal loops have elevated pKa values. The protonation of these adenosines can stabilize the NPSL2 structure through electrostatic interactions. Our study provides fundamental insights into the secondary and tertiary structure of an important RNA hairpin proposed to regulate miR biogenesis.

Predicting Drosha and Dicer Cleavage Sites with DeepMirCut.

MicroRNAs are a class of small RNAs involved in post-transcriptional gene silencing with roles in disease and development. Many computational tools have been developed to identify novel microRNAs. However, there have been no attempts to predict cleavage sites for Drosha from primary sequence, or to identify cleavage sites using deep neural networks. Here, we present DeepMirCut, a recurrent neural network-based software that predicts both Dicer and Drosha cleavage sites. We built a microRNA primary sequence database including flanking genomic sequences for 34,713 microRNA annotations. We compare models trained on sequence data, sequence and secondary structure data, as well as input data with annotated structures. Our best model is able to predict cuts within closer average proximity than results reported for other methods. We show that a guanine nucleotide before and a uracil nucleotide after Dicer cleavage sites on the 3′ arm of the microRNA precursor had a positive effect on predictions while the opposite order (U before, G after) had a negative effect. Our analysis was also able to predict several positions where bulges had either positive or negative effects on the score. We expect that our approach and the data we have curated will enable several future studies.

Somatic Mutations in miRNA Genes in Lung Cancer-Potential Functional Consequences of Non-Coding Sequence Variants.

A growing body of evidence indicates that miRNAs may either drive or suppress oncogenesis. However, little is known about somatic mutations in miRNA genes. To determine the frequency and potential consequences of miRNA gene mutations, we analyzed whole exome sequencing datasets of 569 lung adenocarcinoma (LUAD) and 597 lung squamous cell carcinoma (LUSC) samples generated in The Cancer Genome Atlas (TCGA) project. Altogether, we identified 1091 somatic sequence variants affecting 522 different miRNA genes and showed that half of all cancers had at least one such somatic variant/mutation. These sequence variants occurred in most crucial parts of miRNA precursors, including mature miRNA and seed sequences. Due to our findings, we hypothesize that seed mutations may affect miRNA:target interactions, drastically changing the pool of predicted targets. Mutations may also affect miRNA biogenesis by changing the structure of miRNA precursors, DROSHA and DICER cleavage sites, and regulatory sequence/structure motifs. We identified 10 significantly overmutated hotspot miRNA genes, including the miR-379 gene in LUAD enriched in mutations in the mature miRNA and regulatory sequences. The occurrence of mutations in the hotspot miRNA genes was also shown experimentally. We present a comprehensive analysis of somatic variants in miRNA genes and show that some of these genes are mutational hotspots, suggesting their potential role in cancer.

Enhancement of gene knockdown efficiency by CNNC motifs in the intronic shRNA precursor.

Short hairpin RNAs (shRNAs) expressed from vectors have been used as an effective means of exploiting the RNA interference (RNAi) pathway in mammalian cells. Of several methods to express shRNA, a method of transcribing shRNAs embedded in microRNA precursors has been more widely used than the one that directly expresses shRNA from RNA polymerase III promoters because the microRNA precursor form of shRNA is known to cause lower levels of cytotoxicity and off-target effects than the overexpressed shRNAs from the RNA polymerase III promoters. We study the primary sequence features of microRNA precursors, which enhance their processing into mature form, helps design more potent shRNA precursors embedded in microRNA precursors. We measure the enhancement of gene knockdown efficiency by adding CNNC motifs in the 3' flanking region of shRNA precursor embedded in the human miR-30a microRNA precursor. By systemically adding three CNNC motifs in the 3' flanking region of shRNA precursor, we found that addition of two CNNC motifs saturates their enhanced knockdown ability of shRNA and that the CNNC motif in the + 17 to + 20 from the drosha cleavage site is most important for the shRNA-mediated target gene knock down. We also did see little knockdown of target gene expression by the shRNA precursor lacking CNNC motif. Since genetic studies generally require techniques that could reduce gene expression at different degrees, the findings in this study will allow us to use RNAi for genetic studies of reducing gene expression at different degrees.

RNA Editing of Tumor Suppressor Mir-26a Promotes Malignant Progenitor Propagation in Chronic Myeloid Leukemia

Recent studies demonstrate the importance of post-transcriptional adenosine-to-inosine (A-to-I) RNA editing mediated by adenosine deaminase acting on RNA1 (ADAR1) in normal fetal and adult hematopoiesis. RNA-sequencing studies have shown that elevated levels of the ADAR1 editase has emerged as a dominant driver of cancer progression and therapeutic resistance. Specifically, the deregulation of ADAR1 promotes the transformation of chronic myeloid leukemia (CML) from chronic phase (CP) to a therapy resistant blast crisis (BC) phase. Through the regulation of mRNA and microRNA (miRNA) stability, ADAR1 plays a pivotal role in embryonic development and stem cell regulation. We have previously shown that inflammation-responsive ADAR1 heavily contributes to stem cell differentiation and self-renewal in CML disease progression. Here, we describe a novel role of ADAR1 in cell cycle regulation of BC leukemia cells through regulation of miRNA biogenesis.To investigate the role of ADAR1 in miRNA regulation, we performed miRNome miScript PCR array analysis of 1008 miRNAs in cord blood CD34+ expressing hematopoietic stem and progenitor cells (HSPCs) overexpressing ADAR1 wild type (WT) and A-to-I editing deficient ADAR1 mutant. Overall, a total of 112 miRNAs were significantly differentially expressed following ADAR1 expression with cell cycle identified as the top cellular pathway significantly targeted by miRNAs regulated by ADAR1. Notably, ADAR1 editase activity inhibits the expression of miR-26a-5p, a tumor suppressor miRNA that is frequently downregulated in BC CML. ADAR1 inhibits miR-26a-5p through direct editing of the DROSHA cleavage site of primary miR-26a-5p, preventing miR-26a-5p maturation and processing. In normal hematopoietic progenitors, ADAR1-mediated inhibition of miR-26a results in repression of cyclin-dependent kinase inhibitor 1A (CDKN1A) expression indirectly via suppression of the polycomb repressive complex, enhancer of zeste homolog 2 (EZH2), thereby accelerating cell cycle transit. However, in BC CML progenitors, decreased EZH2 and increased CDKN1A oppose the cell cycle accelerating effect of ADAR1. Moreover, we found that the miR-26a targets a different set of mRNA in BC CML compared to cord blood HSPC and has divergent roles in cell cycle regulation. Lentiviral miR-26a overexpression reduced BC leukemia stem cell (LSC) dormancy in the bone marrow and reverses the functional effects of ADAR1, including inhibition of BC cell proliferation in vivo and impaired LSC self-renewal capacity as measured by colony forming assays.Our finding reveals the effects of ADAR1 in LSC generation through impairing biogenesis of cell cycle regulatory miRNAs. The deregulation of ADAR1 contributes to the malignant reprogramming of progenitors into dormant LSCs that are resistant to therapeutic treatments. Future development of ADAR1 inhibitors may be effective in the elimination of dormant BC CML LSCs that evade tyrosine kinase inhibitors. DisclosuresNo relevant conflicts of interest to declare.

FCLIP-seq for transcriptomic footprinting of dsRNA-binding proteins: Lessons from DROSHA.

CLIP-seq (crosslinking immunoprecipitation and sequencing) is widely used to map the binding sites of a protein of interest on the transcriptome, and generally employs UV to induce the covalent bonds between protein and RNA, which allows stringent washing. However, dsRNA is inefficiently crosslinked by UV, making it difficult to study the interactions between dsRNA binding proteins and their substrates. A dsRNA endoribonuclease DROSHA initiates the maturation of microRNA (miRNA) by cleaving primary miRNA (pri-miRNA). Despite the importance of DROSHA in miRNA maturation and sequence determination, accurate mapping of DROSHA cleavage sites has not been feasible due to rapid processing, modification, and degradation of the cleaved products in cells. Here, we present a high-throughput sequencing method that allows the mapping of in vivo DROSHA cleavage sites at single nucleotide resolution, termed formaldehyde crosslinking, immunoprecipitation, and sequencing (fCLIP-seq). The fCLIP-seq protocol has been improved significantly over the standard CLIP-seq methods by (1) using formaldehyde for efficient and reversible crosslinking, (2) employing polyethylene glycol and adaptors with randomized sequences to enhance ligation efficiency and minimize bias, and (3) performing ligation after elution, which exposes the RNA termini for efficient ligation. fCLIP-seq successfully captures the nascent products of DROSHA, which allows precise mapping of the DROSHA processing sites. Moreover, from the analysis of the distinctive cleavage pattern, we discover previously unknown substrates of DROSHA. fCLIP-seq is a useful tool to obtain transcriptome-wide information on DROSHA activity and can be applied further to investigate other dsRNA-protein interactions.

Comparing miRNA structure of mirtrons and non-mirtrons

BackgroundMicroRNAs proceeds through the different canonical and non-canonical pathways; the most frequent of the non-canonical ones is the splicing-dependent biogenesis of mirtrons. We compare the mirtrons and non-mirtrons of human and mouse to explore how their maturation appears in the precursor structure around the miRNA.ResultsWe found the coherence of the overhang lengths what indicates the dependence between the cleavage sites. To explain this dependence we suggest the 2-lever model of the Dicer structure that couples the imprecisions in Drosha and Dicer. Considering the secondary structure of all animal pre-miRNAs we confirmed that single-stranded nucleotides tend to be located near the miRNA boundaries and in its center and are characterized by a higher mutation rate. The 5′ end of the canonical 5′ miRNA approaches the nearest single-stranded nucleotides what suggests the extension of the loop-counting rule from the Dicer to the Drosha cleavage site. A typical structure of the annotated mirtron pre-miRNAs differs from the canonical pre-miRNA structure and possesses the 1- and 2 nt hanging ends at the hairpin base. Together with the excessive variability of the mirtron Dicer cleavage site (that could be partially explained by guanine at its ends inherited from splicing) this is one more evidence for the 2-lever model. In contrast with the canonical miRNAs the mirtrons have higher snp densities and their pre-miRNAs are inversely associated with diseases. Therefore we supported the view that mirtrons are under positive selection while canonical miRNAs are under negative one and we suggested that mirtrons are an intrinsic source of silencing variability which produces the disease-promoting variants. Finally, we considered the interference of the pre-miRNA structure and the U2snRNA:pre-mRNA basepairing. We analyzed the location of the branchpoints and found that mirtron structure tends to expose the branchpoint site what suggests that the mirtrons can readily evolve from occasional hairpins in the immediate neighbourhood of the 3′ splice site.ConclusionThe miRNA biogenesis manifests itself in the footprints of the secondary structure. Close inspection of these structural properties can help to uncover new pathways of miRNA biogenesis and to refine the known miRNA data, in particular, new non-canonical miRNAs may be predicted or the known miRNAs can be re-classified.

G-Quadruplexes influence pri-microRNA processing

ABSTRACTRNA G-Quadruplexes (G4) have been shown to possess many biological functions, including the regulation of microRNA (miRNA) biogenesis and function. However, their impact on pri-miRNA processing remains unknown. We identified G4 located near the Drosha cleavage site in three distinct pri-miRNAs: pri-mir200c, pri-mir451a, and pri-mir497. The folding of the potential G4 motifs was determined in solution. Subsequently, mutations disrupting G4 folding led to important changes in the mature miRNAs levels in cells. Moreover, using small antisense oligonucleotides binding to the pri-miRNA, it was possible to modulate, either positively or negatively, the mature miRNA levels. Together, these data demonstrate that G4 motifs could contribute to the regulation of pri-mRNA processing, a novel role for G4. Considering that bio-informatics screening indicates that between 9% and 50% of all pri-miRNAs contain a putative G4, these structures possess interesting potential as future therapeutic targets.

Abstract 299: RNA editing enzyme ADAR1 accelerates normal hematopoiesis cell cycle by regulation microRNA biogenesis

Abstract Compelling murine studies demonstrate that adenosine-to-inosine (A-to-I) RNA editing mediated by adenosine deaminase associated with RNA1 (ADAR1) is vital for both fetal and adult hematopoiesis. While genetic ablation of ADAR1 leads to murine embryonic lethality due to severe defects in erythropoiesis, conditional deletion in the hematopoietic system impairs maintenance indicative of cell type and context specific roles for ADAR1 in cell fate specification and self-renewal. By regulating mRNA and microRNA (miRNA) stability, ADAR1 exhibit wide-ranging effects on embryonic development and stem cell regulation. We have previously shown that inflammation-responsive ADAR1 plays important roles in both stem cell differentiation and self-renewal in CML (chronic myeloid leukemia) disease progression. Here, we describe a novel function of ADAR1 in cell cycle regulation of normal hematopoietic stem cell and progenitors (HSPC) by regulation of miRNA biogenesis. Our results demonstrated that ADAR1 induces G0 to G1 phase transition in normal cord blood HSPCs, as demonstrated by elevated expression of Ki67, reduced DiR signal, and enhanced in vivo cord blood engraftment. Cell cycle qRT-qPCR microarray of 84 cell cycle transcripts and whole transcriptome RNA-sequencing analysis of KEGG cell cycle pathway indicate that several cell cycle genes are differentially expression upon overexpression of ADAR1 WT or an A-to-I editing deficient ADAR1 mutant (ADAR1E912A). We previously demonstrated that impaired biogenesis of let-7 miRNAs by ADAR1 WT induces enhanced self-renewal in cord blood CD34+ HSPCs. To determine the miRNA targets of ADAR1-mediated RNA editing, we performed miRNome miScript PCR array of 1008 miRNA candidates in cord blood CD34+ HSPCs overexpressing ADAR1 WT or ADAR1E912A. Total of 263 miRNAs were differentially expressed (142 upregulated and 121 downregulated) by comparing ADAR1 WT to the backbone control. Interestingly, ADAR1E912A mutant also exhibit A-to-I editing independent regulation of miRNAs (307 upregulated and 59 downregulated). We found that the expression of miR-26a-5p, a miRNA frequently downregulated in leukemia, is inhibited by ADAR1-mediated RNA editing. ADAR1 directly binds and edits the DROSHA cleavage site of primary miR-26a transcript, thereby prevent miR26a-5p maturation. Moreover, lentiviral expression of mature miR26-5p reverses the effect of ADAR1 WT, including enhanced CDKN1A expression, inhibition of cord blood proliferation in vivo, as well as reduced HSC self-renewal as measured by colony-formation assay. Our finding suggests carefully regulated A-to-I editing by ADAR1 is essential for the maintenance of proper cell proliferation in HSC. For future study, it will be interesting to investigate if the elevated expression of ADAR1 in CML BC LSC contributes to false regulation of cell cycle that leads to the expansion of malignant leukemia stem cells. Citation Format: Qingfei Jiang, Maria Anna Zipeto, Nathan Delos Santos, Sheldon Morris, Catriona Jamieson. RNA editing enzyme ADAR1 accelerates normal hematopoiesis cell cycle by regulation microRNA biogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 299. doi:10.1158/1538-7445.AM2017-299

Genome-wide Mapping of DROSHA Cleavage Sites on Primary MicroRNAs and Noncanonical Substrates

MicroRNA (miRNA) maturation is initiated by DROSHA, a double-stranded RNA (dsRNA)-specific RNase III enzyme. By cleaving primary miRNAs (pri-miRNAs) at specific positions, DROSHA serves as a main determinant of miRNA sequences and a highly selective gatekeeper for the canonical miRNA pathway. However, the sites of DROSHA-mediated processing have not been annotated, and it remains unclear to what extent DROSHA functions outside the miRNA pathway. Here, we establish a protocol termed "formaldehyde crosslinking, immunoprecipitation, and sequencing (fCLIP-seq)," which allows identification of DROSHA cleavage sites at single-nucleotide resolution. fCLIP identifies numerous processing sites, suggesting widespread end modifications during miRNA maturation. fCLIP also finds many pri-miRNAs that undergo alternative processing, yielding multiple miRNA isoforms. Moreover, we discovered dozens of DROSHA substrates on non-miRNA loci, which may serve as cis-elements for DROSHA-mediated gene regulation. We anticipate that fCLIP-seq could be a general tool for investigating interactions between dsRNA-binding proteins and structured RNAs.

RNA Editing Enzyme ADAR1 Accelerates Normal Hematopoiesis Cell Cycle By Regulating microRNA Biogenesis

Compelling murine studies demonstrate that adenosine-to-inosine (A-to-I) RNA editing mediated by adenosine deaminase associated with RNA1 (ADAR1) is vital for both fetal and adult hematopoiesis. While genetic ablation of ADAR1 editase leads to murine embryonic lethality due to severe defects in erythropoiesis, conditional deletion in the hematopoietic system impairs maintenance indicative of cell type and context specific roles for ADAR1 in cell fate specification and self-renewal. By regulating mRNA and microRNA (miRNA) stability, ADAR1 exhibit wide-ranging effects on embryonic development and stem cell regulation. We have previously shown that inflammation-responsive ADAR1 plays important roles in both stem cell differentiation and self-renewal in CML (chronic myeloid leukemia) disease progression. Here, we describe a novel function of ADAR1 in cell cycle regulation of normal hematopoietic stem cell and progenitors (HSPC) by regulation of miRNA biogenesis.Our results demonstrated that ADAR1 induces G0 to G1 phase transition in normal cord blood HSPCs, as demonstrated by elevated expression of Ki67, reduced DiR signal, and enhanced in vivo cord blood engraftment. Cell cycle qRT-qPCR microarray of 84 cell cycle transcripts and whole transcriptome RNA-sequencing analysis of KEGG cell cycle pathway indicate that several cell cycle genes (CDKN1A, CDKN2A, CCNC, CCND1, BRCA2, etc) are differentially expression upon overexpression of ADAR1 WT or an A-to-I editing deficient ADAR1 mutant (ADAR1E912A). We previously demonstrated that impaired biogenesis of let-7 miRNAs by ADAR1 WT induces enhanced self-renewal in cord blood CD34+ HSPCs. To determine the miRNA targets of ADAR1-mediated RNA editing, we performed miRNome miScript PCR array of 1008 miRNA candidates in cord blood CD34+ HSPCs overexpressing ADAR1 WT or ADAR1E912A. Total of 263 miRNAs were differentially expressed (142 upregulated and 121 downregulated) by comparing ADAR1 WT to the backbone control. Interestingly, ADAR1E912A mutant also exhibit A-to-I editing independent regulation of miRNAs (307 upregulated and 59 downregulated). We found that the expression of miR-26a-5p, a miRNA frequently downregulated in leukemia, is inhibited by ADAR1-mediated RNA editing. ADAR1 directly binds and edits the DROSHA cleavage site of primary miR-26a transcript, thereby prevent miR26a-5p maturation. Moreover, lentiviral expression of mature miR26-5p reverses the effect of ADAR1 WT, including enhanced CDKN1A expression, inhibition of cord blood proliferation in vivo, as well as reduced HSC self-renewal as measured by colony-formation assay.Our finding suggests carefully regulated A-to-I editing by ADAR1 is essential for the maintenance of proper cell proliferation in HSC. For future study, it will be interesting to investigate if the elevated expression of ADAR1 in CML BC LSC contributes to false regulation of cell cycle that leads to the expansion of malignant leukemia stem cells. DisclosuresJamieson:CTI Biopharma: Research Funding; GlaxoSmithKline: Research Funding; Johnson & Johnson: Research Funding.

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.

Sequence features of Drosha and Dicer cleavage sites affect the complexity of isomiRs.

The deep-sequencing of small RNAs has revealed that different numbers and proportions of miRNA variants called isomiRs are formed from single miRNA genes and that this effect is attributable mainly to imprecise cleavage by Drosha and Dicer. Factors that influence the degree of cleavage precision of Drosha and Dicer are under investigation, and their identification may improve our understanding of the mechanisms by which cells modulate the regulatory potential of miRNAs. In this study, we focused on the sequences and structural determinants of Drosha and Dicer cleavage sites, which may explain the generation of homogeneous miRNAs (in which a single isomiR strongly predominates) as well as the generation of heterogeneous miRNAs. Using deep-sequencing data for small RNAs, we demonstrate that the generation of homogeneous miRNAs requires more sequence constraints at the cleavage sites than the formation of heterogeneous miRNAs. Additionally, our results indicate that specific Drosha cleavage sites have more sequence determinants in miRNA precursors than specific cleavage sites for Dicer and that secondary structural motifs in the miRNA precursors influence the precision of Dicer cleavage. Together, we present the sequence and structural features of Drosha and Dicer cleavage sites that influence the heterogeneity of the released miRNAs.

A central role for the primary microRNA stem in guiding the position and efficiency of Drosha processing of a viral pri-miRNA.

Processing of primary microRNA (pri-miRNA) stem-loops by the Drosha-DGCR8 complex is the initial step in miRNA maturation and crucial for miRNA function. Nonetheless, the underlying mechanism that determines the Drosha cleavage site of pri-miRNAs has remained unclear. Two prevalent but seemingly conflicting models propose that Drosha-DGCR8 anchors to and directs cleavage a fixed distance from either the basal single-stranded (ssRNA) or the terminal loop. However, recent studies suggest that the basal ssRNA and/or the terminal loop may influence the Drosha cleavage site dependent upon the sequence/structure of individual pri-miRNAs. Here, using a panel of closely related pri-miRNA variants, we further examine the role of pri-miRNA structures on Drosha cleavage site selection in cells. Our data reveal that both the basal ssRNA and terminal loop influence the Drosha cleavage site within three pri-miRNAs, the Simian Virus 40 (SV40) pri-miRNA, pri-miR-30a, and pri-miR-16. In addition to the flanking ssRNA regions, we show that an internal loop within the SV40 pri-miRNA stem strongly influences Drosha cleavage position and efficiency. We further demonstrate that the positions of the internal loop, basal ssRNA, and the terminal loop of the SV40 pri-miRNA cooperatively coordinate Drosha cleavage position and efficiency. Based on these observations, we propose that the pri-miRNA stem, defined by internal and flanking structural elements, guides the binding position of Drosha-DGCR8, which consequently determines the cleavage site. This study provides mechanistic insight into pri-miRNA processing in cells that has numerous biological implications and will assist in refining Drosha-dependent shRNA design.

Lower and upper stem–single-stranded RNA junctions together determine the Drosha cleavage site

Microprocessor [Drosha-DGCR8 (DiGeorge syndrome critical region gene 8) complex] processing of primary microRNA (pri-miRNA) is the critical first step in miRNA biogenesis, but how the Drosha cleavage site is determined has been unclear. Previous models proposed that the Drosha-DGCR8 complex measures either ~22 nt from the upper stem-single-stranded RNA (ssRNA, terminal loop) junction or ~11 nt from the lower stem-ssRNA junction to determine the cleavage site. Here, using miRNA-offset RNAs to determine the Drosha cleavage site, we show that the Microprocessor measures the distances from both the lower and upper stem-ssRNA junctions to determine the cleavage site in human cells, and optimal distances from both structures are critical to the precision of Drosha processing. If the distances are not optimal, Drosha tends to cleave at multiple sites, which can, in turn, generate multiple 5' isomiRs. Thus, our results also reveal a mechanism of 5' isomiR generation.

Ensemble Analysis of Primary MicroRNA Structure Reveals an Extensive Capacity To Deform near the Drosha Cleavage Site

Most noncoding RNAs function properly only when folded into complex three-dimensional (3D) structures, but the experimental determination of these structures remains challenging. Understanding of primary microRNA (miRNA) maturation is currently limited by a lack of determined structures for nonprocessed forms of the RNA. SHAPE chemistry efficiently determines RNA secondary structural information with single-nucleotide resolution, providing constraints suitable for input into MC-Pipeline for refinement of 3D structure models. Here we combine these approaches to analyze three structurally diverse primary microRNAs, revealing deviations from canonical double-stranded RNA 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 noncanonical structure elements, combine with the necessary single-stranded RNA-double-stranded RNA junction to define the correct Drosha cleavage site.

Expanding the role of Drosha to the regulation of viral gene expression

It is well-appreciated that viruses use host effectors for macromolecular synthesis and as regulators of viral gene expression. Viruses can encode their own regulators, but often use host-encoded factors to optimize replication. Here, we show that Drosha, an endoribonuclease best known for its role in the biogenesis of microRNAs (miRNAs), can also function to directly regulate viral gene expression. Kaposi's Sarcoma-associated Herpesvirus (KSHV) is associated with various tumors, and like all herpesviruses, has two modes of infection, latent and lytic, which are characterized by differential expression of viral gene products. Kaposin B (KapB) is a KSHV-encoded protein associated with cytokine production and cytotoxicity. We demonstrate that in addition to previously known transcriptional mechanisms, differences in Drosha levels contribute to low levels of KapB expression in latency and robust increases in expression during lytic replication. Thus, surprisingly, KSHV modulates Drosha activity differentially depending on the mode of replication. This regulation is dependent on Drosha-mediated cleavage, and KapB transcripts lacking the Drosha cleavage sites express higher levels of KapB, resulting in increased cell death. This work increases the known functions of Drosha and implies that tying viral gene expression to Drosha activity is advantageous for viruses.