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Title: Transcriptome-Wide Cleavage Site Mapping on Cellular mRNAs Reveals Features Underlying Sequence-Specific Cleavage by the Viral Ribonuclease SOX

Abstract

Many viruses express factors that reduce host gene expression through widespread degradation of cellular mRNA. An example of this class of proteins is the mRNA-targeting endoribonuclease SOX from the gamma-herpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV). Previous studies indicated that cleavage of messenger RNAs (mRNA) by SOX occurs at specific locations defined by the sequence of the target RNA, which is at odds with the down-regulation of a large portion of cellular transcripts. In this study, we address this paradox by using high-throughput sequencing of cleavage intermediates combined with a custom bioinformatics-based analysis pipeline to identify SOX cleavage sites across the mRNA transcriptome. These data, coupled with targeted mutagenesis, reveal that while cleavage sites are specific and reproducible, they are defined by a degenerate sequence motif containing a small number of conserved residues rather than a strong consensus sequence. This degenerate element is well represented in both human and KSHV mRNA, and its presence correlates with RNA destabilization by SOX. This represents a new endonuclease targeting strategy, in which use of a degenerate targeting element enables RNA cleavage at specific locations without restricting the range of targets. Furthermore, it shows that strong target selectivity can be achieved without a high degree ofmore » sequence specificity.« less

Authors:
 [1];  [2];  [3]
  1. Tufts Univ., Medford, MA (United States); Tufts Univ. School of Medicine, Boston, MA (United States)
  2. Harvard Univ., Cambridge, MA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  3. Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.; Univ. of California, Berkeley, CA (United States). Dept. of Plant and Microbial Biology
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC); National Institutes of Health (NIH)
OSTI Identifier:
1524004
Grant/Contract Number:  
AC02-05CH11231; NIH CA136367; NIH CA160556
Resource Type:
Accepted Manuscript
Journal Name:
PLoS Pathogens
Additional Journal Information:
Journal Volume: 11; Journal Issue: 12; Journal ID: ISSN 1553-7374
Publisher:
Public Library of Science
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES

Citation Formats

Gaglia, Marta Maria, Rycroft, Chris H., and Glaunsinger, Britt A. Transcriptome-Wide Cleavage Site Mapping on Cellular mRNAs Reveals Features Underlying Sequence-Specific Cleavage by the Viral Ribonuclease SOX. United States: N. p., 2015. Web. https://doi.org/10.1371/journal.ppat.1005305.
Gaglia, Marta Maria, Rycroft, Chris H., & Glaunsinger, Britt A. Transcriptome-Wide Cleavage Site Mapping on Cellular mRNAs Reveals Features Underlying Sequence-Specific Cleavage by the Viral Ribonuclease SOX. United States. https://doi.org/10.1371/journal.ppat.1005305
Gaglia, Marta Maria, Rycroft, Chris H., and Glaunsinger, Britt A. Tue . "Transcriptome-Wide Cleavage Site Mapping on Cellular mRNAs Reveals Features Underlying Sequence-Specific Cleavage by the Viral Ribonuclease SOX". United States. https://doi.org/10.1371/journal.ppat.1005305. https://www.osti.gov/servlets/purl/1524004.
@article{osti_1524004,
title = {Transcriptome-Wide Cleavage Site Mapping on Cellular mRNAs Reveals Features Underlying Sequence-Specific Cleavage by the Viral Ribonuclease SOX},
author = {Gaglia, Marta Maria and Rycroft, Chris H. and Glaunsinger, Britt A.},
abstractNote = {Many viruses express factors that reduce host gene expression through widespread degradation of cellular mRNA. An example of this class of proteins is the mRNA-targeting endoribonuclease SOX from the gamma-herpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV). Previous studies indicated that cleavage of messenger RNAs (mRNA) by SOX occurs at specific locations defined by the sequence of the target RNA, which is at odds with the down-regulation of a large portion of cellular transcripts. In this study, we address this paradox by using high-throughput sequencing of cleavage intermediates combined with a custom bioinformatics-based analysis pipeline to identify SOX cleavage sites across the mRNA transcriptome. These data, coupled with targeted mutagenesis, reveal that while cleavage sites are specific and reproducible, they are defined by a degenerate sequence motif containing a small number of conserved residues rather than a strong consensus sequence. This degenerate element is well represented in both human and KSHV mRNA, and its presence correlates with RNA destabilization by SOX. This represents a new endonuclease targeting strategy, in which use of a degenerate targeting element enables RNA cleavage at specific locations without restricting the range of targets. Furthermore, it shows that strong target selectivity can be achieved without a high degree of sequence specificity.},
doi = {10.1371/journal.ppat.1005305},
journal = {PLoS Pathogens},
number = 12,
volume = 11,
place = {United States},
year = {2015},
month = {12}
}

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Figures / Tables:

Fig 1 Fig 1: PARE experimental procedure and peak finding analysis pipeline. A) Diagram of the PARE procedure. B) Schematic of PyDegradome analysis approach, which uses read counts in a control sample to generate a table of thresholds to compare test sample counts to. The table lists thresholds for a particular user-definedmore » confidence level and for a range of ratios between control and test samples. The applicable ratio for each position is computed by multiplying a user-defined multiplicative factor by the ratio of total read counts for the exon in test vs. control samples, thus accounting for variation in RNA levels and total mapped reads. Read counts in test sample at each position must exceed the threshold to be identified as part of a peak. C) Example of plot of read counts (5’ end only) for 200 nt surrounding a SOX cut site identified by PyDegradome within the 3’–most exon of the LIMD1 NM_014240 transcript in the four samples. Note that y-axis has a logarithmic scale. This example shows the expected distribution for a cut site followed by exonucleolytic degradation due to residual Xrn1 activity or to the action of another nuclease, with the highest read count at cut site and decaying counts at following positions. Positions referred to as “peak” and “cut site” in the text are marked on the graph. D) Flow chart of steps used to defined SOX cut sites used for further analyses.« less

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      Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.