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Re-evaluation of the roles of DROSHA, Exportin 5, and DICER in microRNA biogenesis

ABSTRACT: Biogenesis of canonical microRNAs (miRNAs) involves multiple steps: nuclear processing of primary miRNA (pri-miRNA) by DROSHA, nuclear export of precursor miRNA (pre-miRNA) by Exportin 5 (XPO5), and cytoplasmic processing of pre-miRNA by DICER. To gain a deeper understanding of the contribution of each of these maturation steps, we deleted DROSHA, XPO5, and DICER in the same human cell line, and analyzed their effects on miRNA biogenesis. Canonical miRNA production was completely abolished in DROSHA-deleted cells while we detected a few DROSHA-independent miRNAs including three previously unidentified noncanonical miRNAs (miR-7706, miR-3615, and miR-1254). In contrast to DROSHA knockout, many canonical miRNAs were still detected without DICER albeit at markedly reduced levels. In the absence of DICER, pre-miRNAs are loaded directly onto AGO and trimmed at the 3′ end, yielding miRNAs from the 5′ strand (5p miRNAs). Interestingly, in XPO5 knockout cells, most miRNAs are affected only modestly, suggesting that XPO5 is necessary but not critical for miRNA maturation. Our study demonstrates an essential role of DROSHA and an important contribution of DICER in the canonical miRNA pathway, and reveals that the function of XPO5 can be complemented by alternative mechanisms. Thus, this study allows us to understand differential contributions of key biogenesis factors, and provides with valuable resources for miRNA research.

ORGANISM(S): Homo sapiens

PROVIDER: GSE77989 | GEO | 2016/03/17

SECONDARY ACCESSION(S): PRJNA312310

REPOSITORIES: GEO

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Similar Datasets

Re-evaluation of the roles of DROSHA, Exportin 5, and DICER in microRNA biogenesis

Project description:Biogenesis of canonical microRNAs (miRNAs) involves multiple steps: nuclear processing of primary miRNA (pri-miRNA) by DROSHA, nuclear export of precursor miRNA (pre-miRNA) by Exportin 5 (XPO5), and cytoplasmic processing of pre-miRNA by DICER. To gain a deeper understanding of the contribution of each of these maturation steps, we deleted DROSHA, XPO5, and DICER in the same human cell line, and analyzed their effects on miRNA biogenesis. Canonical miRNA production was completely abolished in DROSHA-deleted cells while we detected a few DROSHA-independent miRNAs including three previously unidentified noncanonical miRNAs (miR-7706, miR-3615, and miR-1254). In contrast to DROSHA knockout, many canonical miRNAs were still detected without DICER albeit at markedly reduced levels. In the absence of DICER, pre-miRNAs are loaded directly onto AGO and trimmed at the 3â² end, yielding miRNAs from the 5â² strand (5p miRNAs). Interestingly, in XPO5 knockout cells, most miRNAs are affected only modestly, suggesting that XPO5 is necessary but not critical for miRNA maturation. Our study demonstrates an essential role of DROSHA and an important contribution of DICER in the canonical miRNA pathway, and reveals that the function of XPO5 can be complemented by alternative mechanisms. Thus, this study allows us to understand differential contributions of key biogenesis factors, and provides with valuable resources for miRNA research. Two independent sequencing experiments (set 1 and set 2, respectively) were performed using 9 samples.

2016-03-17 | E-GEOD-77989 | biostudies-arrayexpress

XPO5 is required for miRNA biogenesis and promotes primary miRNA processing independent of RanGTP

Project description:XPO5 mediates nuclear export of miRNA hairpin precursors in a RanGTP-dependent manner. However, the requirement of XPO5 for global miRNA biogenesis and mammalian development and XPO5-associated RNA species are not determined. Here we show that XPO5 is required for mouse embryonic development and morphogenesis of skin and brain. Loss of XPO5 compromises the biogenesis of most miRNAs and that leads to severe developmental defects. Surprisingly, XPO5 not only associates with miRNA hairpin precursors but also pervasively binds to double-stranded RNA (dsRNA) regions found in many cellular RNAs and some clustered pri-miRNAs. The binding of XPO5 to miR-17~92 pri-miRNAs is RanGTP-independent. Pre-incubation with XPO5 enhances the processing efficiency of the DROSHA/DGCR8 microprocessor. Together, our studies demonstrate the requirement of XPO5 for miRNA biogenesis and mouse development, reveal an unexpected role of XPO5 for recognizing and facilitating the nuclear cleavage of clustered pri-miRNAs and identify numerous cellular RNAs as novel XPO5 subtracts.

2020-02-19 | GSE111964 | GEO

Amyotrophic Lateral Sclerosis-associated Mutants of SOD1 Modulate miRNA Biogenesis through Aberrant Interactions with Exportin 5

Project description:Mutations in SOD1 (Superoxide Dismutase 1) gene are associated with amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease. By employing ascorbate peroxidase (APEX)-based proximity labeling, coupled with LC-MS/MS analysis, we uncovered 37 and 28 proteins exhibiting higher abundance in the proximity proteomes of SOD1G85R and SOD1G93A, respectively, than that of the wild-type SOD1. Immunoprecipitation followed by western blot analysis confirmed the preferential binding of one of these proteins, exportin 5 (XPO5), toward the two mutants of SOD1 over its wild-type counterpart. In line with the established function of XPO5 in pre-miRNA transport, we observed diminished nucleocytoplasmic transport of pre-miRNAs in cells with ectopic expression of the two SOD1 mutants over those expressing the wild-type counterpart. On the other hand, RT-qPCR results revealed significant elevations in mature miRNA in cells expressing the two SOD1 mutants, which are attributed to diminished inhibitory effect of XPO5 on Dicer-mediated cleavage of pre-miRNA to mature miRNA. Together, our chemoproteomic approach led to the revelation of a novel mechanism through which ALS-associated mutants of SOD1 perturb miRNA biogenesis, i.e., through aberrant binding toward XPO5.

2024-01-26 | PXD035452 | Pride

A compendium of RNA-binding proteins that regulate microRNA biogenesis

Project description:During microRNA-biogenesis, stem-loop structured precursors are processed in two endonucleolytic steps, giving rise to mature microRNAs (miRNAs). This process is not only regulated at the level of transcription but also posttranscriptionally by RNA-binding proteins (RBPs). Here we have used a proteomics-based pull-down approach to map and characterize the interactome of 72 pre-miRNAs in eleven cell lines. We identify over 150 RBPs that interact specifically with distinct pre-miRNAs. To demonstrate their functional relevance, we used RNAi knockdown and CRISPR/Cas-mediated knockout experiments and analyzed changes in miRNA levels. Indeed, a large number of the investigated candidates have effects on miRNA-processing under our experimental conditions, including several C3H-zinc-finger proteins. Ultimately, we show that TRIM71/Lin41 is a potent regulator of miR-29a processing and its inactivation directly affects miR-29a targets. In summary, we provide an extended database of RBPs that interact with pre-miRNAs in different cell types helping to elucidate posttranscriptional regulation of miRNA biogenesis on a global scale.

2017-04-26 | PXD004193 | Pride

A quantitative map of human primary microRNA processing sites

Project description:Maturation of canonical microRNA (miRNA) is initiated by DROSHA that cleaves the primary transcript (pri-miRNA). Over 1,800 miRNA loci are annotated in humans, but it remains largely unknown if and at which sites the pri-miRNAs are cleaved by DROSHA. Here we performed in vitro processing on a full set of human pri-miRNAs (miRBase v21) followed by sequencing. This comprehensive profiling enabled us to classify miRNAs based on DROSHA-dependence and map their cleavage sites with respective processing efficiency measures. Only 758 pri-miRNAs are confidently processed by DROSHA, while the majority may be non-canonical or false entries. Analyses of the DROSHA-dependent pri-miRNAs show key cis-elements for processing. We observe widespread alternative processing as well as unproductive cleavage events such as “nick” or “inverse” processing. SRSF3 is a broad-acting auxiliary factor modulating alternative processing and suppressing unproductive processing. The profiling data and methods developed in this study will allow systematic analyses of miRNA regulation.

2021-07-28 | PXD025767 | Pride

Sequencing of Argonaute-bound miRNA/mRNA hybrids reveals regulation of the unfolded protein response by microRNA-320a

Project description:MicroRNAs (miRNA) are short non-coding RNAs widely implicated in development, gene regulation, and disease progression. Most miRNAs utilize the RNase III enzymes Drosha and Dicer for biogenesis in animals. One notable exception is the RNA polymerase II transcription start sites (TSS) miRNAs whose biogenesis requires Dicer but not Drosha. The functional importance of the TSS-miRNA biogenesis pathway has remained uncertain due to their unelucidated targetomes. To better understand the function of TSS-miRNAs, we applied a modified Crosslinking, Ligation, and Sequencing of Hybrids on Argonaute (Ago-qCLASH) to identify the targets for TSS-miRNAs in HCT116 colorectal cancer cells with or without Drosha knockout (KO). We observed that miR-320a hybrids dominate in TSS-miRNA hybrids identified by Ago-qCLASH. Targets for miR-320a are enriched in the eIF2 signaling pathway, a downstream component of the unfolded protein response. Consistently, in miR-320a mimic- and inhibitor- transfected cells, differentially expressed genes are enriched in the eIF2 signaling pathway. Within the Ago-qCLASH data, we identified the endoplasmic reticulum (ER) chaperone Calnexin as a direct miR-320a target, thus connecting miR-320a to the unfolded protein response. During ER stress, but not amino acid deprivation, miR-320a up-regulates ATF4, a critical transcription factor for resolving ER stress. Our study helps to elucidate the targetome of the TSS-miRNAs in colorectal cancer cells and establishes miR-320a as a regulator of unfolded protein response.

2021-06-23 | GSE164634 | GEO

Global identification of target recognition and cleavage by the Microprocessor in human ESCs.

Project description:The Microprocessor plays an essential role in canonical miRNA biogenesis by facilitating cleavage of stem-loop structures in primary transcripts to yield pre-miRNAs. Although miRNA biogenesis has been extensively studied through biochemical and molecular genetic approaches, it has yet to be addressed to what extent the current miRNA biogenesis models hold true in intact cells. To address the issues of in vivo recognition and cleavage by the Microprocessor, we investigate RNAs that are associated with DGCR8 and Drosha by using immunoprecipitation coupled with next-generation sequencing. Here, we present global protein-RNA interactions with unprecedented sensitivity and specificity. Our data indicate that precursors of canonical miRNAs and miRNA-like hairpins are the major substrates of the Microprocessor. As a result of specific enrichment of nascent cleavage products, we are able to pinpoint the Microprocessor-mediated cleavage sites per se at single-nucleotide resolution. Unexpectedly, a 2-nt 3M-bM-^@M-^Y overhang invariably exists at the ends of cleaved bases instead of nascent pre-miRNAs. Besides canonical miRNA precursors, we find that two novel miRNA-like structures embedded in mRNAs are cleaved to yield pre-miRNA-like hairpins, uncoupled from miRNA maturation. Our data provide a framework for in vivo Microprocessor-mediated cleavage and a foundation for experimental and computational studies on miRNA biogenesis in living cells. CLIP-seq for DGCR8 and Drosha, RIP-seq for DGCR8, sequencing of AGO2-assocated miRNA

2014-10-02 | E-GEOD-61979 | biostudies-arrayexpress

Endogenous shRNAs, siRNAs, and Other Microprocessor-independent, Dicer-dependent Small RNAs in Mouse ES Cells

Project description:Canonical microRNAs (miRNAs) require two processing steps: the first by the Microprocessor, a complex of DGCR8 and Drosha, and the second by Dicer. dgcr8delta/delta mouse embryonic stem cells (mESCs) have less severe phenotypes than dicer1delta/delta mESCs, suggesting a physiological role for Microprocessor-independent, Dicer-dependent small RNAs. To identify these small RNAs with unusual biogenesis, we performed high-throughput sequencing from wild type, dgcr8delta/delta, and dicer1delta/delta mESCs. Several of the DGCR8-independent, Dicer-dependent RNAs were non-canonical miRNAs. These derived from mirtrons and a newly identified subclass of miRNA precursors, which appears to be the endogenous counterpart of short hairpin RNAs (shRNAs). Our analyses also revealed endogenous siRNAs resulting from Dicer cleavage of long hairpins, the vast majority of which originated from one genomic locus with tandem, inverted short interspersed nuclear elements (SINEs). Our results extend the known diversity of mammalian small-RNA generating pathways and show that mammalian siRNAs exist in tissues outside of oocytes.

2008-09-10 | GSE12521 | GEO

Endogenous shRNAs, siRNAs, and Other Microprocessor-independent, Dicer-dependent Small RNAs in Mouse ES Cells

2008-09-09 | E-GEOD-12521 | biostudies-arrayexpress

Microarray profiling reveals broad and differential effects of RBM3 on miRNA expression.

Project description:MicroRNAs (miRNAs) are a family of short, noncoding RNAs that regulate translation of mRNAs by mechanisms involving the binding of complementary sequences. The influence of miRNAs on the proteome and cellular events is extensive as they regulate an estimated 60% of the transcriptome and play key roles in differentiation, plasticity, circadian rhythm, immunity, and disease. The post-transcriptional biogenesis of most miRNAs involves a sequential cleavage process mediated by RNase III family enzymes. Primary transcripts (pri-miRNAs) are first cleaved by Drosha in the nucleus to yield ~70nt hairpin precursors (pre-miRNAs). These intermediates are transported to the cytoplasm where ~22mer dsRNAs are excised by Dicer. Typically, one strand of the dsRNAs is inserted as a mature miRNA into the RNA-induced silencing complex (RISC), which contains members of the Argonaute (Ago) protein family that contribute to translational regulation. Recent studies indicate that some RNA-binding proteins (RNA-BPs) can regulate discrete processing steps, differentially blocking or promoting the formation of specific miRNAs to control cellular proliferation and differentiation. Elegant examples of this mechanism include the attenuation of let-7 biogenesis in embryonic stem cells by the pluripotency factor LIN28, and the selective enhancement of miR-18a biogenesis from a polycistronic transcript by hnRNPA1. Mature miRNA expression profiles in B104 cells were measured after siRNA-mediated knockdown of RBM3 in the B104 neuronal cell line. Mock treated B104 cells were used as a control. Experiments were replicated in duplicates.

2011-11-10 | E-GEOD-33519 | biostudies-arrayexpress

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