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Global analysis of truncated RNA ends reveals novel insights into ribosome stalling in plants

ABSTRACT: High-throughput approaches for profiling the 5' ends of RNA degradation intermediates on a genome-wide scale are frequently applied in the validation of cleavage sites guided by microRNAs (miRNAs). However, the complexity of the RNA degradome beyond miRNA targets is currently largely unclear which limits the application of RNA degradome. Here, we provide multiple lines of evidence to show that the plant RNA degradome contains in vivo ribosome-protected mRNA fragments. We observed a 3-nt periodicity and a bias toward the translational frame in the global analysis of 5' truncated mRNA ends mapped to the coding sequence (CDS) of Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa) and soybean (Glycine max). In addition, predominant 5' termini of RNA degradation intermediates separated by a length equal to a ribosome-protected mRNA fragment were evident in some conserved peptide upstream open reading frames (uORFs) of Arabidopsis and rice. Through the analysis of RNA degradome data, we uncovered novel uORFs and CDS regions potentially associated with stacked ribosomes in Arabidopsis. Furthermore, the analysis of RNA degradome data suggested that the binding of Arabidopsis ARGONAUTE7 to a non-cleavable target site of miR390 might directly hinder ribosome movement. This work demonstrates an alternative use of RNA degradome data in the study of ribosome stalling.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE77549 | GEO | 2016/11/30

SECONDARY ACCESSION(S): PRJNA310776

REPOSITORIES: GEO

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Arabidopsis mRNA decay landscape shaped by XRN 5′-3′ exoribonucleases

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2022-09-06 | GSE205190 | GEO

RNA degradomes reveal substrates and importance for dark and nitrogen stress responses of Arabidopsis XRN4

Project description:Application of Parallel Analysis of RNA Ends (PARE), an RNA degradome approach to identify substrates of Arabidopsis XRN4. The xrn4 mutants overaccumulate decay intermediates from multiple mRNA turnover pathways. This material is based on work supported by the National Science Foundation [under Grant Numbers MCB1021636 and MCB1817764 to P.J.G.]

2019-08-13 | GSE119706 | GEO

Degradome sequencing reveals endogenous small RNA targets in rice (Oryza sativa L. ssp. indica)

Project description:MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) regulate gene expression in eukaryotes. Plant miRNAs modulate their targets mainly via messenger RNA (mRNA) cleavage. Small RNA targets have been extensively investigated in Arabidopsis using computational prediction, experimental validation, and degradome sequencing. However, small RNA targets are largely unknown in rice (Oryza sativa). Here, we report global identification of small RNA targets using high throughput degradome sequencing in the rice indica cultivar 93-11 (Oryza sativa L. ssp. indica). 177 transcripts targeted by total of 87 unique miRNAs were identified. Of targets for the conserved miRNAs between Arabidopsis and rice, transcription factors comprise around 70% (58 in 82), indicating that these miRNAs act as masters of gene regulatory nodes in rice. In contrast, non-conserved miRNAs targeted diverse genes which provide more complex regulatory networks. In addition, 5 AUXIN RESPONSE FACTORS (ARF) cleaved by the TAS3 derived ta-siRNAs were also detected. A total of 40 sRNA targets were further validated via RNA ligase-mediated 5M-bM-^@M-^Y rapid amplification of cDNA ends (RLM 5M-bM-^@M-^Y-RACE). Our degradome results present a detailed sRNA-target interaction atlas, which provides a guide for the study of the roles of sRNAs and their targets in rice. The degradome sequence of Young inflorescences from Oryza sativa L. ssp. indica (93-11) was sequenced

2010-01-01 | E-GEOD-19050 | biostudies-arrayexpress

Degradome sequencing reveals endogenous small RNA targets in rice (Oryza sativa L. ssp. indica)

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2010-01-01 | GSE19050 | GEO

Translation of upstream open reading frames in a model of neuronal differentiation

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2018-09-07 | GSE119615 | GEO

Widespread EJC footprints in the RNA degradome mark mRNA degradation before steady-state translation

Project description:Exon junction complexes (EJCs) are deposited to mRNAs during splicing and displaced from mRNAs by ribosomes in the pioneer round of translation. The understanding of EJC-bound mRNA degradation before steady-state translation has been limited to nonsense-mediated mRNA decay (NMD) due to a lack of suitable methodologies. Here, we show that RNA degradome data of Arabidopsis, rice, worm and human cells all exhibit a predominant accumulation of 5′ monophosphate (5′P) ends in the canonical EJC region. Inhibition of 5′-3′ exoribonuclease activity and overexpression of an EJC disassembly factor in Arabidopsis reduced the 5′P ends accumulating in the EJC region, supporting the notion that these 5′P ends are in vivo EJC footprints. Hundreds of Arabidopsis NMD targets possess evident EJC footprints, validating their degradation during the pioneer round of translation. In addition to premature termination codons, plant microRNAs can also direct the degradation of EJC-bound mRNAs. However, the production of EJC footprints from NMD but not microRNA targets is dependent on an NMD factor, SMG7. Together, our finding demonstrating in vivo EJC footprinting unravels the composition of the RNA degradome, and provides a new avenue for studying NMD and other mechanisms, such as microRNAs, targeting EJC-bound mRNAs for degradation before steady-state translation.

2020-02-02 | GSE118215 | GEO

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2015-06-04 | GSE63120 | GEO

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Project description:MicroRNA (miRNA)-guided target RNA expression is vital for a wide variety of biological processes in eukaryotes. The integration of miRNAs in diverse biological networks relies upon the confirmation of their RNA targets. Most miRNA targets in Arabidopsis are validated, but those in rice are yet to be characterized. To identify transcriptome-wide small RNA targets in rice, we generated 20-nt small cDNA library and obtained nearly 40 million reads. Sequence analysis yielded 11,552,007 unique reads that can be perfectly mapped to the rice genome. Sequence analysis not only found homologous targets for conserved miRNAs but also many novel targets. Besides miRNA atregts, the rice degradome contained fragments derived from MIRNA precursors. A closer inspection of these fragments revealed a unique pattern distinct from siRNA producing loci. This attribute can serve as one of the ancillary criteria for separating miRNAs from siRNAs in plants. Keywords: high throughout sequencing of rice degradome Identify transcriptome-wide small RNA targets in rice

2010-05-03 | E-GEOD-17398 | biostudies-arrayexpress

Widespread Co-translational RNA Decay Reveals Ribosome Dynamics

Project description:It is generally assumed that mRNAs undergoing translation are protected from decay. Here, we show that mRNAs are, in fact, co-translationally degraded. This is a widespread and conserved process affecting most genes, where 5′–3′ transcript degradation follows the last translating ribosome, producing an in vivo ribosomal footprint. By sequencing the ends of 5′ phosphorylated mRNA degradation intermediates, we obtain a genome-wide drug-free measurement of ribosome dynamics. We identify general translation termination pauses in both normal and stress conditions. In addition, we describe novel codon-specific ribosomal pausing sites in response to oxidative stress that are dependent on the RNase Rny1. Our approach is simple and straightforward and does not require the use of translational inhibitors or in vitro RNA footprinting that can alter ribosome protection patterns. RNA samples were quantified according to their 5’modification. Samples were quantified using 2 alternative methods. 5PSeq (measures 5’P RNAs) and 5PSeq-fragmented (measures the sequencing bias present in any sample by randomly fragmenting RNA, re-phosphorilating the 5’ends and subjecting the sample to 5PSeq; it is used as a negative control). Different strains of S. cerevisiae and S. pombe samples were analyzed. Cells were grown both in rich media (YPD or YES) and synthetic defined media (SD). Cells were subjected to different treatments such as inhibition of translation elongation with cyclohexamide (CHX), oxidative stress (0.2 mM H2O2) or inhibition of histidine biosyntesys (3-AT; 100mM 3-amino-1,2,4-triazole). S. cerevisiae samples grown in rich media were also analyzed after CHX treatment and sucrose fractionation into monosome and polyribosome fractionation. All samples were analyzed in biological duplicates.

2015-06-04 | E-GEOD-63120 | biostudies-arrayexpress

Transcriptome-wide identification of small RNA targets in rice

2010-05-04 | GSE17398 | GEO

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