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Series GSE61398 Query DataSets for GSE61398
Status Public on Dec 01, 2014
Title Direct identification of endogenous SMG6 targets and a preferred motif spanning SMG6 cleavage sites by parallel analysis of RNA ends in human cells
Organism Homo sapiens
Experiment type Expression profiling by high throughput sequencing
Summary In metazoans, the endoribonuclease SMG6 is thought to cleave many endogenous mRNAs targeted for nonsense-mediated mRNA decay (NMD). However, most evidence as to the identity of endogenous SMG6 substrates is indirect, and little is known about their cleavage sites. Here, we report the efficacy of an RNA degradome approach called parallel analysis of RNA ends (PARE) for identifying NMD intermediates in human cells. By specifically sequencing the 5’ ends of intermediates dependent on SMG6 and the critical NMD factor UPF1, hundreds of endogenous transcripts that are direct targets of SMG6 have been revealed. A preferred sequence motif spanning most SMG6 cleavage sites has been identified and validated by mutational analysis. For many SMG6 substrates, depletion of SMG6 leads to decapping of the RNAs. These findings provide key insights into NMD and targeting by SMG6 while also demonstrating the potential of PARE for analyzing other ribonucleases with diverse endogenous substrates.
Overall design Genome-wide degradome sequencing and mRNA profiling was done following the knockdown of SMG6 and UPF1. SPARE libraries targeting a sequence motif surrounding the identified cleavage site were also deeply sequenced on an Illumina platform.
PARE is a method of degradome analysis that captures and deeply sequences the 5' monophosphorylated ends of polyadenylated RNAs (see.German 2009 Nat Protoc 4, 356-362).  C-PARE libraries capture capped, polyadenlyated RNAs by first dephosphorylating the RNA, then decapping using tobacco acid pyrophosphatase and constructing PARE libraries, and will be described in the forthcoming paper.  SPARE captures the 5' monophosphorylated ends of specific RNAs using transcript-specific primers (see Schapire 3013 Methods 64:283-291).
Contributor(s) Schmidt S, Foley P, Belesco J, Green P
Citation(s) 25429978
Submission date Sep 13, 2014
Last update date May 15, 2019
Contact name Pamela J Green
Organization name University of Delaware
Department Delaware Biotechnology Institute
Street address 15 Innovation Way
City Newark
State/province DE
ZIP/Postal code 19711
Country USA
Platforms (1)
GPL16791 Illumina HiSeq 2500 (Homo sapiens)
Samples (36)
GSM1503892 siGL2+siXRN1 Bio1 PARE
GSM1503893 siSMG6+siXRN1 Bio1 PARE
GSM1503894 siUPF1+siXRN1 Bio1 PARE
BioProject PRJNA260967
SRA SRP047097

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Supplementary file Size Download File type/resource
GSE61398_RAW.tar 490.6 Mb (http)(custom) TAR (of TXT)
GSE61398_isoform_expression.txt.gz 7.5 Mb (ftp)(http) TXT
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Raw data are available in SRA
Processed data provided as supplementary file

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