title: Identification of SMG6 cleavage sites and a preferred RNA cleavage motif by global analysis of endogenous NMD targets in human cells.
abstract: In metazoans, cleavage by the endoribonuclease SMG6 is often the first degradative event in non-sense-mediated mRNA decay (NMD). However, the exact sites of SMG6 cleavage have yet to be determined for any endogenous targets, and most evidence as to the identity of SMG6 substrates is indirect. Here, we use Parallel Analysis of RNA Ends to specifically identify the 5' termini of decay intermediates whose production is dependent on SMG6 and the universal NMD factor UPF1. In this manner, the SMG6 cleavage sites in hundreds of endogenous NMD targets in human cells have been mapped at high resolution. In addition, a preferred sequence motif spanning most SMG6 cleavage sites has been discovered and validated by mutational analysis. For many SMG6 substrates, depletion of SMG6 resulted in the accumulation of decapped transcripts, an effect indicative of competition between SMG6-dependent and SMG6-independent NMD pathways. These findings provide key insights into the mechanisms by which mRNAs targeted by NMD are degraded.
authors.affiliation: Delaware Biotechnology Institute and Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19711, USA.
authors.forename: Skye A
authors.initials: SA
authors.lastname: Schmidt
authors.affiliation: Kimmel Center for Biology and Medicine at the Skirball Institute and Department of Microbiology, New York University School of Medicine, New York, NY 10016, USA.
authors.forename: Patricia L
authors.initials: PL
authors.lastname: Foley
authors.affiliation: Delaware Biotechnology Institute and Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19711, USA.
authors.forename: Dong-Hoon
authors.initials: DH
authors.lastname: Jeong
authors.affiliation: Delaware Biotechnology Institute and Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19711, USA.
authors.forename: Linda A
authors.initials: LA
authors.lastname: Rymarquis
authors.affiliation: Department of Life Science, Hallym University, Chuncheon, Gangwon, Republic of Korea.
authors.forename: Francis
authors.initials: F
authors.lastname: Doyle
authors.affiliation: Department of Life Science, Hallym University, Chuncheon, Gangwon, Republic of Korea.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Tenenbaum
authors.affiliation: Kimmel Center for Biology and Medicine at the Skirball Institute and Department of Microbiology, New York University School of Medicine, New York, NY 10016, USA joel.belasco@med.nyu.edu.
authors.forename: Joel G
authors.initials: JG
authors.lastname: Belasco
authors.affiliation: Delaware Biotechnology Institute and Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19711, USA green@dbi.udel.edu.
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Green
mesh_terms: D006367:HeLa Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D059365:Nonsense Mediated mRNA Decay; D059372:Nucleotide Motifs; D059367:RNA Cleavage; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D017423:Sequence Analysis, RNA; D019098:Telomerase / Q000378:metabolism*; D004722:Endoribonucleases
chemical_list: D012333:RNA, Messenger; D019098:Telomerase; C512261:SMG6 protein, human; D004722:Endoribonucleases