title: Role of Rck-Pat1b binding in assembly of processing-bodies.
abstract: The DEAD box RNA helicase Rck and the scaffold protein Pat1b participate in controlling gene expression at the post-transcriptional level by suppressing mRNA translation and promoting mRNA decapping. In addition, both proteins are required for the assembly of processing (P)-bodies, cytoplasmic foci that contain stalled mRNAs and numerous components of the mRNA decay machinery. The C-terminal RecA-like domain of Rck interacts with the N-terminal acidic domain of Pat1b. Here, we identified point mutations in human Rck and Pat1b that prevent the two proteins from binding to each other. By analyzing interaction-deficient mutants in combination with knockdown and rescue strategies in human HeLa cells, we found that Pat1b assembles P-bodies and suppresses expression of tethered mRNAs in the absence of Rck binding. In contrast, Rck requires the Pat1b-binding site in order to promote P-body assembly and associate with the decapping enzyme Dcp2 as well as Ago2 and TNRC6A, two core components of the RNA-induced silencing complex. Our data indicate that P-body assembly occurs in a step-wise manner, where Rck participates in the initial suppression of mRNA translation, whereas Pat1b in a second step triggers P-body assembly and promotes mRNA decapping.
authors.affiliation: Helmholtz Junior Research Group Posttranscriptional Control of Gene Expression, German Cancer Research Center DKFZ--ZMBH Alliance, Heidelberg, Germany.
authors.forename: Sevim
authors.initials: S
authors.lastname: Ozgur
authors.forename: Georg
authors.initials: G
authors.lastname: Stoecklin
mesh_terms: D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism*; D019143:Evolution, Molecular; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D017354:Point Mutation; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering
chemical_list: D004268:DNA-Binding Proteins; C526510:PATL1 protein, human; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C030052:mRNA decapping enzymes; D004722:Endoribonucleases; C094683:DDX6 protein, human; D053487:DEAD-box RNA Helicases
keywords: PatL1/Pat1b; RNA helicase; RNA-induced silencing complex; Rck/p54; decapping; mRNA degradation; processing body; translation suppression
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