Structural Basis for pri-miRNA Recognition by Drosha

Jin, Wei; Wang, Jia; Liu, Chao-Pei; Wang, Hongwei; Xu, Rui-Ming

doi:10.1016/j.molcel.2020.02.024

Cited by 78 publications

(75 citation statements)

References 41 publications

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“…These authors described both sequence motifs located around the miRNA precursor hairpins and assessed their contribution to the miRNA maturation process [9]. Additional surveys further reported other motifs affecting the expression of miRNAs [10] and analyzed the structural specifications of RNA-protein interactions between miRNAs and protein subunits in the Microprocessor complex [77–79], as well as the influence of such processing motifs in miRNA prediction analyses [80]. Particularly relevant was the study by Fernandez et al (2017) [13], where they described a mutation in the apical loop of hsa-miR-30c (G/A) that creates a steric disruption of the pri-miRNA folding structure of the hairpin, hence creating a bulge around the CNNC motif that facilitates the SRSF3 factor accessibility to the RNA sequence.…”

Section: Discussionmentioning

confidence: 99%

Variability in porcine microRNA genes and its association with mRNA expression and lipid phenotypes

Mármol-Sánchez¹,

Guan

Quintanilla

et al. 2020

Preprint

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25 Background 26 Mature microRNAs (miRNAs) play an important role in repressing the expression of a 27 wide range of protein coding transcripts by promoting their degradation or inhibiting 28 their translation into functional proteins. The presence of segregating polymorphisms 29 inside miRNA loci and their corresponding 3'UTR binding sites might disrupt canonical 30 conserved miRNA-mRNA pairing, thus modifying gene expression patterns. 31 Results 32We aimed to investigate the variability of miRNA genes and their putative binding sites 33 by analyzing whole-genome sequences from 120 pigs and wild boars from Europe and 34 Asia. In total, 285 SNPs residing within miRNA loci were detected. From these, 221 35 were located in precursor regions, whereas 52 and 12 mapped to mature and seed 36 regions, respectively. Moreover, a total of 109,724 polymorphisms were identified in 37 predicted 7mer-m8 miRNA binding sites within porcine 3'UTRs. A principal 38 components analysis revealed a clear genetic divergence between Asian and European 39 samples, which was particularly strong for 3'UTR sequences. We also observed that 40 miRNA genes show reduced polymorphism compared with other non-miRNA regions. 41To assess the potential consequences of miRNA polymorphisms, we sequenced the 42 genomes of 5 Duroc pigs and, by doing so, we identified 15 miRNA SNPs that were 43 genotyped in the offspring (N = 345) of the five boars. Association analyses between 44 miRNA SNPs and hepatic and muscle microarray data allowed us to identify 4 45 polymorphisms displaying significant associations. Particularly interesting was the 46 rs319154814 polymorphism (G/A), located in the apical loop of the ssc-miR-326 47 3 precursor sequence. This polymorphism is predicted to cause a subtle hairpin 48 rearrangement that improves the accessibility to processing enzymatic factors. 49 Conclusions 50 Porcine miRNA genes show a reduced variability, particularly in the seed region which 51 plays a critical role in miRNA binding. Although it is generally assumed that SNPs 52 mapping to the seed region are the ones with the strongest consequences on mRNA 53 expression, we show that a SNP mapping to the apical region of ssc-miR-326 is 54 associated with the mRNA expression of several of its predicted targets. This result 55 suggests that porcine miRNA variability mapping within and outside the seed region 56 could have important regulatory effects on gene expression. 57 58 59 60 61 62 63 64 65 66 67 4 Background 68 Mature microRNA transcripts (miRNAs) are short (~22 nt) non-coding RNAs 69 which play an essential role in the regulation of gene expression [1]. During the 70 biogenesis of miRNAs, one strand of the miRNA duplex binds to the guide-strand 71 channel of an Argonaute protein forming an miRNA-induced silencing complex 72 (miRISC) with the ability of repressing mRNA expression through binding to specific 73 3'UTR target sites [2]. In postembryonic cells, this repressor mechanism mainly acts by 74 destabilizing the mRNA through decapping and poly(A)-tail...

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Section: Discussionmentioning

confidence: 99%

Variability in porcine microRNA genes and its association with mRNA expression and lipid phenotypes

Mármol-Sánchez¹,

Guan

Quintanilla

et al. 2020

Preprint

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“…The primary transcript is processed into shRNA by the Microprocessor, which is composed of one Drosha molecule and two DGCR8 molecules. DGCR8 interacts with the apical loop and stem by its heme-binding domain and dsRNA-binding domains, respectively, allowing efficient and accurate processing (Nguyen et al, 2015;Kwon et al, 2016;Jin et al, 2020;Partin et al, 2020). As a result, the Microprocessor measures 10−11 bp from the basal single-stranded RNA and double-stranded (ds)RNA junction of the primary transcript and cleaves to release the shRNA.…”

Section: Common Shrna Expression Methodologymentioning

confidence: 99%

Short Hairpin RNAs for Strand-Specific Small Interfering RNA Production

Sheng

Flood

Xie

2020

Front. Bioeng. Biotechnol.

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RNA interference (RNAi) is an effective mechanism for inhibiting gene expression at the post-transcriptional level. Expression of a messenger RNA (mRNA) can be inhibited by a ∼22-nucleotide (nt) small interfering (si)RNA with the corresponding reverse complementary sequence. Typically, a duplex of siRNA, composed of the desired siRNA and a passenger strand, is processed from a short hairpin RNA (shRNA) precursor by Dicer. Subsequently, one strand of the siRNA duplex is associated with Argonaute (Ago) protein for RNAi. Although RNAi is widely used, the off-target effect induced by the passenger strand remains a potential problem. Here, based on current understanding of endogenous precursor microRNA (pre-miRNA) hairpins, called Ago-shRNA and m 7 G-capped pre-miRNA, we discuss the principles of shRNA designs that produce a single siRNA from one strand of the hairpin.

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“…The heme cofactor is important for proper orientation of pri-miRNA hairpins in Microprocessor and for preferentially processing of distinct groups of pri-miRNAs, including those containing the UGU motif 24,25,26 . Most recently, cryo-EM structures of Microprocessor in complex with pri-miRNAs have revealed an extensive protein-RNA interface, but pri-miRNA apical junctions and loops and the Rhed domain could not be resolved 27,28 .…”

Section: Introductionmentioning

confidence: 99%

Structures of microRNA-precursor apical junctions and loops reveal non-canonical base pairs important for processing

Shoffner

Peng

Guo

2020

Preprint

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Metazoan pri-miRNAs and pre-miRNAs fold into characteristic hairpins that are recognized by the processing machinery. Essential to the recognition of these miR-precursors are their apical junctions where double-stranded stems meet single-stranded hairpin loops. Little is known about how apical junctions and loops fold in three-dimensional space. Here we developed a scaffold-directed crystallography method and determined the structures of eight human miR-precursor apical junctions and loops. Six structures contain non-canonical base pairs stacking on top of the hairpin stem. U-U pair contributes to thermodynamic stability in solution and is highly enriched at human miR-precursor apical junctions. Our systematic mutagenesis shows that U-U is among the most efficiently processed variants.The RNA-binding heme domain of pri-miRNA-processing protein DGCR8 binds longer loops more tightly and non-canonical pairs at the junction appear to modulate loop length. Our study provides structural and biochemical bases for understanding miR-precursors and molecular mechanisms of microRNA maturation.

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Structural Basis for pri-miRNA Recognition by Drosha

Cited by 78 publications

References 41 publications

Variability in porcine microRNA genes and its association with mRNA expression and lipid phenotypes

Variability in porcine microRNA genes and its association with mRNA expression and lipid phenotypes

Short Hairpin RNAs for Strand-Specific Small Interfering RNA Production

Structures of microRNA-precursor apical junctions and loops reveal non-canonical base pairs important for processing

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