Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

The menu of features that define primary microRNAs and enable de novo design of microRNA genes

ABSTRACT: MicroRNAs (miRNAs) are small regulatory RNAs processed from stem-loop regions of primary transcripts (pri-miRNAs), with the choice of stem-loops for initial processing largely determining what becomes a miRNA. To identify sequence and structural features influencing this choice, we determined cleavage efficiencies of >50,000 variants of three human pri-miRNAs, focusing on the regions intractable to previous high-throughput analyses. Our analyses revealed a mismatched motif in the basal stem region, a preference for maintaining or improving base-pairing throughout the remainder of the stem, and a narrow stem-length preference of 35±1 base pairs. Incorporating these features with previously identified features, including three primary-sequence motifs, yielded a unifying model defining mammalian pri-miRNAs, in which motifs help orient processing and increase efficiency, with the presence of more motifs compensating for structural defects. This model enables generation of artificial pri-miRNAs, designed de novo, without reference to any natural sequence, yet processed more efficiently than natural pri-miRNAs.

ORGANISM(S): synthetic construct Homo sapiens

PROVIDER: GSE67937 | GEO | 2015/09/24

SECONDARY ACCESSION(S): PRJNA281255

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

The menu of features that define primary microRNAs and enable de novo design of microRNA genes

Project description:MicroRNAs (miRNAs) are small regulatory RNAs processed from stem-loop regions of primary transcripts (pri-miRNAs), with the choice of stem-loops for initial processing largely determining what becomes a miRNA. To identify sequence and structural features influencing this choice, we determined cleavage efficiencies of >50,000 variants of three human pri-miRNAs, focusing on the regions intractable to previous high-throughput analyses. Our analyses revealed a mismatched motif in the basal stem region, a preference for maintaining or improving base-pairing throughout the remainder of the stem, and a narrow stem-length preference of 35±1 base pairs. Incorporating these features with previously identified features, including three primary-sequence motifs, yielded a unifying model defining mammalian pri-miRNAs, in which motifs help orient processing and increase efficiency, with the presence of more motifs compensating for structural defects. This model enables generation of artificial pri-miRNAs, designed de novo, without reference to any natural sequence, yet processed more efficiently than natural pri-miRNAs. Three major experiments are included in the submitted data. 1) Pools of synthetic DNA containing barcodes and pri-miRNA variants were sequenced to infer the barcode-pri-miRNA linkages, and the pri-miRNA variants were assayed for their cleavage efficiency ("selection"), which can be quantified by comparing their representations in the input and after cleavage. 2) Artificial miRNAs produced in HEK293T cells were sequenced. 3) Transcriptomes of artificial miRNA-expressed cells were sequenced to quantify mRNA changes.

2015-09-24 | E-GEOD-67937 | biostudies-arrayexpress

Non-canonical RNA substrates of Drosha lack many of the conserved features found in primary microRNA stem-loops

Project description:The RNase III enzyme Drosha has a central role in microRNA (miRNA) biogenesis, where it required to release the stem-loop intermediate from primary (pri)-miRNA transcripts. However, it can also cleave stem-loops embedded within messenger (m)RNAs. This destabilizes the mRNA causing target gene repression and has been found to occur primarily in stem cells. While pri-miRNA stem-loops have been extensively studied, such non-canonical substrates of Drosha has yet to characterized in detail. In this study, we employed high-throughput sequencing to capture all polyA-tailed RNAs that are cleaved by Drosha in mouse embryonic stem cells (ESCs). We then compared the features of non-canonical versus miRNA stem-loop substrates. First, these mRNA substrates are less efficiently processed than miRNA stem-loops. Sequence and structural analyses revealed that these substrates are also less stable and more likely to fold into alternative structures than miRNA stem-loops. Moreover, they lack the sequence and structural motifs found in miRNAs stem-loops that are required for precise cleavage. Notably, we discovered a non-canonical Drosha substrate that is cleaved in an inverse manner, which is a process that is normally inhibited by features in miRNA stem-loops. Our study thus provides valuable insights into the recognition of non-canonical targets by Drosha.

2024-03-27 | GSE228299 | GEO

Microprocessor recruitment to elongating RNA Polymerase II is required for differential expression of microRNAs

Project description:The cellular abundance of mature microRNAs (miRNAs) is dictated by the efficiency of nuclear processing of primary miRNA transcripts (pri-miRNAs) into pre-miRNA intermediates. The Microprocessor complex of Drosha and DGCR8 carries this out, but it has been unclear what controls Microprocessor's differential processing of various pri-miRNAs. Here, we show that Drosophila DGCR8 (Pasha) directly associates with the C terminal domain of the RNA polymerase II elongation complex when it is phosphorylated by the Cdk9 kinase (pTEFb). When association is blocked by loss of Cdk9 activity, a global change in pri-miRNA processing is detected. Processing of pri-miRNAs with a UGU sequence motif in their apical junction domain increases, while processing of pri-miRNAs lacking this motif decreases. Therefore, phosphorylation of RNA polymerase II recruits Microprocessor for co-transcriptional processing of non-UGU pri-miRNAs that would otherwise be poorly processed. In contrast, UGU-positive pri-miRNAs are robustly processed by Microprocessor independent of RNA polymerase association.

2017-09-15 | GSE103234 | GEO

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

High-throughput in vitro DROSHA processing on human pri-miRNA variants

Project description:The in vitro high-throughput human pri-miRNA processing assays were conducted to check whether mismatches and wobble base pairs in the upper stem of pri-miRNAs affects the DROSHA cleavage.

2020-02-28 | GSE142140 | GEO

Structural atlas of human primary microRNAs generated by SHAPE-MaP

Project description:MicroRNA (miRNA) maturation is critically dependent on structural features of primary transcripts (pri-miRNAs). However, the scarcity of determined pri-miRNA structures has limited our understanding of miRNA maturation. Here we employed SHAPE-MaP, a high-throughput RNA structure probing method, to unravel the secondary structures of 476 high-confidence human pri-miRNAs. Our SHAPE-based structures diverge substantially from those inferred solely from computation, particularly in the apical loop and basal segments, underlining the need for experimental data in RNA structure prediction. By comparing the structures with high-throughput processing data, we determined the optimal structural features of pri-miRNAs. The sequence determinants are influenced substantially by their structural contexts. Moreover, we identified an element termed the bulged GWG motif (bGWG) with a 3′ bulge in the lower stem, which promotes processing. Our structure-function mapping better annotates the determinants of pri-miRNA processing and offers practical implications for designing small hairpin RNAs and predicting the impacts of miRNA mutations.

2024-01-31 | GSE254361 | GEO

The effect of mismatches and wobble base pairs in the upper stem of pri-miRNAs on DROSHA cleavage using small RNA sequencing

Project description:The experiments were conducted to check whether mismatches and wobble base pairs in the upper stem of pri-miRNAs affects the DROSHA cleavage and thereby miRNA biogenesis.

2020-02-28 | GSE142136 | GEO

The SHAPE-MaP experiment to identify RNA structures of pri-mir-142 variants

Project description:MicroRNAs (miRNAs) are short non-coding RNAs that play essential roles in RNA silencing and gene regulation. The human Microprocessor (MP) is the key factor to initiate miRNA biogenesis by cleaving primary microRNAs (pri-miRNAs). However, the Microprocessor alone cannot precisely and efficiently cleave all pri-miRNAs; thus, it requires cofactors to assist its cleavage. SRSF3 interacts with CNNC in pri-miRNAs, enhancing the MP cleavage. However, it is unknown if SRSF3 can function with other non-CNNC motifs and if secondary structure might influce CNNC function. In addition, function of SRSF7, a paralog of SRSF3, in the SR proteins family, in miRNA biogenesis is largely unknown. In this study, by conducting the high-throughput pri-miRNA cleavage assays for the MP with SRSF3 or SRSF7 and randomized pri-miRNAs, we discovered that SRSF7 also stimulate MP clevage. Futhermore, we found that both SRSF3 and SRSF7 can function with some non-CNNC motifs and with CNNC motifs with certain secondary structures. Furthermore, we also demonstrated that SRSF7 and SRSF3 governed the cleavage sites of the Microprocessor in human cells. Our findings disclose the roles SRSF7 in miRNA biogenesis, demonstrate a compresenhive moleucalr mechanism of SFSF3 and SRSF7 in enhancing cleavage of MP and described in more detail the RNA-binding features of SRSF7 and SRSF3.

2023-01-23 | GSE221996 | GEO

Structural basis for pre-miRNA-31 biogenesis

Project description:MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate protein gene expression post-transcriptionally. By base pairing with target mRNAs, miRNAs triggers mRNA degradation or translational suppression[1-4]. Abnormal miRNAs levels are associated with cancers, diabetes, neurological and other diseases[5-8]. RNA polymerase II transcribes primary miRNA (pri-miR) in the nucleus and pri-miRs are subsequently processed by Microprocessor, which is composed of Drosha and DiGeorge syndrome critical region 8 (DGCR8) proteins, to generate precursor miRNAs (pre-miRs). Pre-miRs are exported from the nucleus to the cytoplasm in a GTP-dependent manner by Exportin-5. In the cytoplasm, a pre-miRs are further processed by Dicer to generate 21-22 nt mature miRNA[4, 9]. Argonaute (Ago) protein loads one of mature miRNAs into protein and forms miRNA-induced silencing complex, which is responsible for mRNA degradation or translational suppression[2, 4].

2023-04-24 | GSE230167 | GEO

The high-throughput human pri-miRNA processing assays

Project description:Human Microprocessor cleaves pri-miRNAs to initiate miRNA biogenesis. The accuracy and efficiency of Microprocessor cleavage ensure appropriate miRNA sequence and expression and thus its proper gene regulation. However, Microprocessor cleaves many pri-miRNAs incorrectly, so it requires assistance from its many cofactors. For example, SRSF3 enhances Microprocessor cleavage by interacting with the CNNC motif in pri-miRNAs. However, whether SRSF3 can function with other motifs and/or requires the motifs in a certain secondary structure is unknown. In addition, the function of SRSF7 (a paralog of SRSF3) in miRNA biogenesis still needs to be discovered. Here, we demonstrated that SRSF7 could stimulate Microprocessor cleavage. In addition, by conducting high-throughput pri-miRNA cleavage assays for Microprocessor and SRSF7 or SRSF3, we demonstrated that SRSF7 and SRSF3 function with the CRC and CNNC motifs, adopting certain secondary structures. In addition, SRSF7 and SRSF3 affect the Microprocessor cleavage sites in human cells. Our findings demonstrate the roles of SRSF7 in miRNA biogenesis and provide a comprehensive view of the molecular mechanism of SRSF7 and SRSF3 in enhancing Microprocessor cleavage.

2023-01-23 | GSE215790 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data