Dataset Information

Improved computational analysis of ribosome dynamics from 5'P degradome data using fivepseq.

ABSTRACT: In eukaryotes, 5'-3' co-translation degradation machinery follows the last translating ribosome providing an in vivo footprint of its position. Thus, 5' monophosphorylated (5'P) degradome sequencing, in addition to informing about RNA decay, also provides information regarding ribosome dynamics. Multiple experimental methods have been developed to investigate the mRNA degradome; however, computational tools for their reproducible analysis are lacking. Here, we present fivepseq: an easy-to-use application for analysis and interactive visualization of 5'P degradome data. This tool performs both metagene- and gene-specific analysis, and enables easy investigation of codon-specific ribosome pauses. To demonstrate its ability to provide new biological information, we investigate gene-specific ribosome pauses in Saccharomyces cerevisiae after eIF5A depletion. In addition to identifying pauses at expected codon motifs, we identify multiple genes with strain-specific degradation frameshifts. To show its wide applicability, we investigate 5'P degradome from Arabidopsis thaliana and discover both motif-specific ribosome protection associated with particular developmental stages and generally increased ribosome protection at termination level associated with age. Our work shows how the use of improved analysis tools for the study of 5'P degradome can significantly increase the biological information that can be derived from such datasets and facilitate its reproducible analysis.

SUBMITTER: Nersisyan L

PROVIDER: S-EPMC7685019 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Improved computational analysis of ribosome dynamics from 5'P degradome data using fivepseq.

Nersisyan Lilit L Ropat Maria M Pelechano Vicent V

NAR genomics and bioinformatics 20201124 4

In eukaryotes, 5'-3' co-translation degradation machinery follows the last translating ribosome providing an <i>in vivo</i> footprint of its position. Thus, 5' monophosphorylated (5'P) degradome sequencing, in addition to informing about RNA decay, also provides information regarding ribosome dynamics. Multiple experimental methods have been developed to investigate the mRNA degradome; however, computational tools for their reproducible analysis are lacking. Here, we present fivepseq: an easy-to ...[more]

PMID: 33575643

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Project description:BackgroundPost-transcriptional gene regulation is one of the critical layers of overall gene expression programs and microRNAs (miRNAs) play an indispensable role in this process by guiding cleavage on the messenger RNA targets. The transcriptome-wide cleavages on the target transcripts can be identified by analyzing the degradome or PARE or GMUCT libraries. However, high-throughput sequencing of PARE or degradome libraries using Illumina platform, a widely used platform, is not so straightforward. Moreover, the currently used degradome or PARE methods utilize MmeI restriction site in the 5' RNA adapter and the resulting fragments are only 20-nt long, which often poses difficulty in distinguishing between the members of the same target gene family or distinguishing miRNA biogenesis intermediates from the primary miRNA transcripts belonging to the same miRNA family. Consequently, developing a method which can generate longer fragments from the PARE or degradome libraries which can also be sequenced easily using Illumina platform is ideal.ResultsIn this protocol, 3' end of the 5'RNA adaptor of TruSeq small RNA library is modified by introducing EcoP15I recognition site. Correspondingly, the double-strand DNA (dsDNA) adaptor sequence is also modified to suit with the ends generated by the restriction enzyme EcoP15I. These modifications allow amplification of the degradome library by primer pairs used for small RNA library preparation, thus amenable for sequencing using Illumina platform, like small RNA library.ConclusionsDegradome library generated using this improved protocol can be sequenced easily using Illumina platform, and the resulting tag length is ~ 27-nt, which is longer than the MmeI generated fragment (20-nt) that can facilitate better accuracy in validating target transcripts belonging to the same gene family or distinguishing miRNA biogenesis intermediates of the same miRNA family. Furthermore, this improved method allows pooling and sequencing degradome libraries and small RNA libraries simultaneously using Illumina platform.

Dataset Information

Improved computational analysis of ribosome dynamics from 5'P degradome data using fivepseq.

Publications

Improved computational analysis of ribosome dynamics from 5'P degradome data using fivepseq.

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