Ubiquitous mRNA decay fragments in E. coli redefine the functional transcriptome
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ABSTRACT:
PROVIDER: PRJNA781857 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR16981271.fastq.gz | Fastqsanger.gz | |||
| SRR16981274.fastq.gz | Fastqsanger.gz | |||
| SRR16981275.fastq.gz | Fastqsanger.gz | |||
| SRR16981276.fastq.gz | Fastqsanger.gz | |||
| SRR16981277.fastq.gz | Fastqsanger.gz |
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Project description:Bacterial mRNAs have short life cycles, in which transcription is rapidly followed by translation and degradation within seconds to minutes. The resulting diversity of mRNA molecules across different life-cycle stages impacts their functionality but has remained unresolved. Here we quantitatively map the 3' status of cellular RNAs in Escherichia coli during steady-state growth and report a large fraction of molecules (median>60%) that are fragments of canonical full-length mRNAs. The majority of RNA fragments are decay intermediates, whereas nascent RNAs contribute to a smaller fraction. Despite the prevalence of decay intermediates in total cellular RNA, these intermediates are underrepresented in the pool of ribosome-associated transcripts and can thus distort quantifications and differential expression analyses for the abundance of full-length, functional mRNAs. The large heterogeneity within mRNA molecules in vivo highlights the importance in discerning functional transcripts and provides a lens for studying the dynamic life cycle of mRNAs.
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Project description:XRN 5′-3′ exoribonucleases play crucial roles in the control of RNA processing, quality, and quantity in eukaryotes. Although genome-wide profiling of RNA decay fragments is now feasible, how XRNs shape the plant mRNA degradome remains elusive. Here, we profiled and analyzed the RNA degradomes of the Arabidopsis wild type and mutants with defects in XRN activity. Deficiency of nuclear XRN3 or cytoplasmic XRN4 but not nuclear XRN2 activity largely altered Arabidopsis mRNA decay profiles. In addition to the primary XRN4 substrates derived from decapping and microRNA-directed slicing, terminating ribosome- and exon junction complex-protected fragments produced from XRN4-mediated cytoplasmic decay also represent the most abundant decay intermediates of Arabidopsis mRNAs. Short excised linear introns and cleaved pre-mRNA fragments downstream of polyadenylation sites were polyadenylated and stabilized in the xrn3 mutant, demonstrating the function of XRN3 in the removal of cleavage remnants from pre-mRNA processing. Further analysis of stabilized XRN3 substrates confirmed that polyadenylation cleavage frequently occurs after an adenosine. An increase in decay intermediates with 5′ ends upstream of a consensus motif in the xrn4 mutant suggests an endonucleolytic cleavage mechanism targeting the 3′ untranslated region of many Arabidopsis mRNAs. However, analysis of decay fragments stabilized in the xrn4 mutant indicated that, except for microRNA-directed slicing, endonucleolytic cleavage events in the coding sequence might rarely result in major decay intermediates. Together, the results of this study reveal major substrates and products of nuclear and cytoplasmic XRNs along Arabidopsis transcripts and provide a basis for precise interpretation of RNA degradome data.
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