Biogenesis of a young, 22-nt microRNA in Phaseoleae species by precursor-programmed uridylation
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ABSTRACT:
PROVIDER: PRJNA464063 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7124411.fastq.gz | Fastqsanger.gz | |||
| SRR7124412.fastq.gz | Fastqsanger.gz | |||
| SRR7124413.fastq.gz | Fastqsanger.gz | |||
| SRR7124414.fastq.gz | Fastqsanger.gz | |||
| SRR7124415.fastq.gz | Fastqsanger.gz |
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Project description:Phased, secondary siRNAs (phasiRNAs) represent a class of small RNAs in plants generated via distinct biogenesis pathways, predominantly dependent on the activity of 22 nt miRNAs. Most 22 nt miRNAs are processed by DCL1 from miRNA precursors containing an asymmetric bulge, yielding a 22/21 nt miRNA/miRNA* duplex. Here we show that miR1510, a soybean miRNA capable of triggering phasiRNA production from numerous NB-LRRs, previously described as 21 nt in its mature form, primarily accumulates as a 22 nt isoform via monouridylation. We demonstrate that in Arabidopsis, this uridylation is performed by HESO1. Biochemical experiments showed that the 3' terminus of miR1510 is only partially 2'-O-methylated, because of the terminal mispairing in the miR1510/miR1510* duplex that inhibits HEN1 activity in soybean. miR1510 emerged in the Phaseoleae ~41 to 42 MYA with a conserved precursor structure yielding a 22 nt monouridylated form, yet a variant in mung bean is processed directly in a 22 nt mature form. This analysis of miR1510 yields two observations: (1) plants can utilize post-processing modification to generate abundant 22 nt miRNA isoforms to more efficiently regulate target mRNA abundances; (2) comparative analysis demonstrates an example of selective optimization of precursor processing of a young plant miRNA.
2018-07-06 | GSE114075 | GEO
Project description:Phased, secondary siRNAs (phasiRNAs) represent a class of small RNAs in plants generated via distinct biogenesis pathways, predominantly dependent on the activity of 22-nt miRNAs. Most 22-nt miRNAs are processed by DCL1 from miRNA precursors containing an asymmetric bulge, yielding a 22/21-nt miRNA/miRNA* duplex. Here we show that miR1510, a soybean miRNA capable of triggering phasiRNA production from numerous nucleotide-binding leucine-rich repeat (NB-LRRs), previously described as 21 nt in its mature form, primarily accumulates as a 22-nt isoform via monouridylation. We demonstrate that, in Arabidopsis, this uridylation is performed by HESO1. Biochemical experiments showed that the 3' terminus of miR1510 is only partially 2'-O-methylated because of the terminal mispairing in the miR1510/miR1510* duplex that inhibits HEN1 activity in soybean. miR1510 emerged in the Phaseoleae ?41-42 million years ago with a conserved precursor structure yielding a 22-nt monouridylated form, yet a variant in mung bean is processed directly in a 22-nt mature form. This analysis of miR1510 yields two observations: (i) plants can utilize postprocessing modification to generate abundant 22-nt miRNA isoforms to more efficiently regulate target mRNA abundances; and (ii) comparative analysis demonstrates an example of selective optimization of precursor processing of a young plant miRNA.
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Project description:A key step in microRNA (miRNA) biogenesis is the processing of a primary precursor RNA by the microprocessor into a precursor miRNA (pre-miRNA) intermediate. In plants, little is known about the processes that act on pre-miRNAs to influence miRNA biogenesis. Here, we performed 3’ RACE-seq to profile pre-miRNA 3’ ends in Arabidopsis. 3’ end heterogeneity was prevalent and the three microprocessor components promoted 3’ end precision. Extensive cytidylation and uridylation of precise and imprecise pre-miRNA 3’ ends were uncovered. The nucleotidyl transferase HESO1 uridylated pre-miRNAs in vitro and was responsible for most pre-miRNA uridylation in vivo. HESO1, NTP6 and NTP7 contribute to pre-miRNA cytidylation. Tailing of pre-miRNAs tended to restore trimmed pre-miRNAs to intact lengths to promote further processing. In addition, HESO1-mediated uridylation led to the degradation of some imprecisely processed pre-miRNAs. Thus, we uncovered widespread cytidylation and uridylation of pre-miRNAs and demonstrated diverse functions of pre-miRNA tailing in plants.
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Project description:Terminal uridylyl transferases (TUTs) function as integral regulators of microRNA (miRNA) biogenesis by modifying the end structure of precursor miRNA (pre-miRNA). Using biochemistry and deep sequencing techniques, we here investigate the mechanism how human TUT7 recognizes and uridylates pre-miRNAs. We show that the overhang of a pre-miRNA is the key structural element that TUT7 and its paralogues, TUT4 and TUT2, recognize. For group II pre-miRNAs which have a 1 nt 3’ overhang, TUT7 restores the canonical end structure (2 nt 3’ overhang) by mono-uridylation, and thereby promotes miRNA biogenesis. Interestingly, once the 3’ end is receded into the stem (3’ trimmed pre-miRNAs such as Ago-cleaved-pre-miRNA), TUT7 effectively generates an oligo-U tail that consequently leads to degradation. Our single-molecule study further suggests that a distributive mode is employed for both pathways, but the overhang length determines the frequency of TUT7-RNA interaction. Our results explain how TUT7 and TUT4 differentiate pre-miRNA species and reveal a role for TUT7 and TUT4 in the oligo-uridylation and removal of defective pre-miRNAs. HeLa cells were knocked down of control or TUT2/4/7, then total RNAs were prepared for RNA-seq
2015-04-30 | E-GEOD-64482 | biostudies-arrayexpress
Project description:Terminal uridylyl transferases (TUTs) function as integral regulators of microRNA (miRNA) biogenesis by modifying the end structure of precursor miRNA (pre-miRNA). Using biochemistry and deep sequencing techniques, we here investigate the mechanism how human TUT7 recognizes and uridylates pre-miRNAs. We show that the overhang of a pre-miRNA is the key structural element that TUT7 and its paralogues, TUT4 and TUT2, recognize. For group II pre-miRNAs which have a 1 nt 3’ overhang, TUT7 restores the canonical end structure (2 nt 3’ overhang) by mono-uridylation, and thereby promotes miRNA biogenesis. Interestingly, once the 3’ end is receded into the stem (3’ trimmed pre-miRNAs such as Ago-cleaved-pre-miRNA), TUT7 effectively generates an oligo-U tail that consequently leads to degradation. Our single-molecule study further suggests that a distributive mode is employed for both pathways, but the overhang length determines the frequency of TUT7-RNA interaction. Our results explain how TUT7 and TUT4 differentiate pre-miRNA species and reveal a role for TUT7 and TUT4 in the oligo-uridylation and removal of defective pre-miRNAs.
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Project description:Prevalent cytidylation and uridylation of precursor miRNAs in Arabidopsis
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Project description:Small non-coding RNA biogenesis typically involves cleavage of structured precursors by RNase III-like endonucleases. However, guide RNAs that direct U-insertion/deletion mRNA editing in mitochondria of trypanosomes maintain 5â² triphosphate characteristic of transcription start site and possess U-tail indicative of 3â² processing and uridylation. Here, we identified a protein complex composed of RET1 TUTase and 3â²-5â² DSS1 exonuclease, and three additional subunits. This complex, termed mitochondrial 3â² processome (MPsome), is responsible for primary uridylation of ~800-nt gRNA precursors, their processive degradation to a mature length of 50-60 nt, and secondary U-tail addition. Both strands of gRNA gene are transcribed giving rise to sense and antisense precursors of similar size. Head-to-head hybridization of these transcripts blocks symmetrical 3â²-5â² degradation at the fixed distance from the double-stranded region. Together, our findings suggest a model in which gRNA is derived from the 5â² extremity of a primary molecule by uridylation-induced, antisense transcription-controlled exonucleolytic degradation. 1. we first sequenced guide RNA precursor (Gel-fractioned total cellular RNA 600-1500nt was used) transcripts from three replicates to study the their tail features and also validate observation of sense/antisense accumulation upon perturbation of pre-processing complex based on few cases. 2. we then sequenced mitochondrial small RNA and built a reference for small RNAs using our custom algorithm. We then took the mitochondrial small RNA data and uncovered the sense/antisense pair. 3. We then used CLIP-Seq data to investigate in vivo binding sites and, together with RNA IP-Seq data to understand what determine the relative abundance of sense and antisense pair of the duplex. 4. We used sequenced small mitochondrial RNA in different RNAi experiments (For RNAi experiments, 30-70nt RNA fraction was gel-isolated from total cellular RNAs) to understand which protein will affect the Utail length in mature small mitochondrial RNA.
2016-02-08 | E-GEOD-71292 | biostudies-arrayexpress
Project description:Small non-coding RNA biogenesis typically involves cleavage of structured precursors by RNase III-like endonucleases. However, guide RNAs that direct U-insertion/deletion mRNA editing in mitochondria of trypanosomes maintain 5′ triphosphate characteristic of transcription start site and possess U-tail indicative of 3′ processing and uridylation. Here, we identified a protein complex composed of RET1 TUTase and 3′-5′ DSS1 exonuclease, and three additional subunits. This complex, termed mitochondrial 3′ processome (MPsome), is responsible for primary uridylation of ~800-nt gRNA precursors, their processive degradation to a mature length of 50-60 nt, and secondary U-tail addition. Both strands of gRNA gene are transcribed giving rise to sense and antisense precursors of similar size. Head-to-head hybridization of these transcripts blocks symmetrical 3′-5′ degradation at the fixed distance from the double-stranded region. Together, our findings suggest a model in which gRNA is derived from the 5′ extremity of a primary molecule by uridylation-induced, antisense transcription-controlled exonucleolytic degradation.
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Project description:Uridylation occurs pervasively on mRNAs in mammals, yet its mechanism and significance remain unknown. Here we identify TUT4 and TUT7 (also known as ZCCHC11 and ZCCHC6, respectively) as the enzymes that uridylate mRNAs. Uridylation readily occurs on deadenylated mRNAs that are not associated with poly(A) binding protein (PABPC1) in cells. Consistently, purified TUT4 and TUT7 (TUT4/7) selectively uridylate RNAs with short A tails (< ~25 nt) while PABPC1 antagonizes uridylation of polyadenylated mRNAs in vitro. In cells depleted of TUT4/7, the vast majority of mRNAs lose the U tails, and their half-lives are extended. Suppression of mRNA decay factors leads to the accumulation of uridylated mRNAs. In line with this, microRNA induces uridylation of its targets, and TUT4/7 is required for enhanced decay of microRNA targets. Our study explains the mechanism underlying selective uridylation of deadenylated mRNAs, and demonstrates a fundamental role of the U tail as a molecular mark for global mRNA decay.
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Project description:Uridylation occurs pervasively on mRNAs in mammals, yet its mechanism and significance remain unknown. Here we identify TUT4 and TUT7 (also known as ZCCHC11 and ZCCHC6, respectively) as the enzymes that uridylate mRNAs. Uridylation readily occurs on deadenylated mRNAs that are not associated with poly(A) binding protein (PABPC1) in cells. Consistently, purified TUT4 and TUT7 (TUT4/7) selectively uridylate RNAs with short A tails (< ~25 nt) while PABPC1 antagonizes uridylation of polyadenylated mRNAs in vitro. In cells depleted of TUT4/7, the vast majority of mRNAs lose the U tails, and their half-lives are extended. Suppression of mRNA decay factors leads to the accumulation of uridylated mRNAs. In line with this, microRNA induces uridylation of its targets, and TUT4/7 is required for enhanced decay of microRNA targets. Our study explains the mechanism underlying selective uridylation of deadenylated mRNAs, and demonstrates a fundamental role of the U tail as a molecular mark for global mRNA decay.
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