Human Pat1b Connects Deadenylation with mRNA Decapping and Controls the Assembly of Processing Bodies

Abstract

In eukaryotic cells, degradation of many mRNAs is initiated by removal of the poly(A) tail followed by decapping and 5′-3′ exonucleolytic decay. Although the order of these events is well established, we are still lacking a mechanistic understanding of how deadenylation and decapping are linked. In this report we identify human Pat1b as a protein that is tightly associated with the Ccr4-Caf1-Not deadenylation complex as well as with the Dcp1-Dcp2 decapping complex. In addition, the RNA helicase Rck and Lsm1 proteins interact with human Pat1b. These interactions are mediated via at least three independent domains within Pat1b, suggesting that Pat1b serves as a scaffold protein. By tethering Pat1b to a reporter mRNA, we further provide evidence that Pat1b is also functionally linked to both deadenylation and decapping. Finally, we report that Pat1b strongly induces the formation of processing (P) bodies, cytoplasmic foci that contain most enzymes of the RNA decay machinery. An amino-terminal region within Pat1b serves as an aggregation-prone domain that nucleates P bodies, whereas an acidic domain controls the size of P bodies. Taken together, these findings provide evidence that human Pat1b is a central component of the RNA decay machinery by physically connecting deadenylation with decapping.

Supplemental material for this article may be found at http://mcb.asm.org/.

We thank Jochen Kreth, Julia Luther, Oksana Seibert, and Gizem Olmezer for technical assistance, Sahil Sharma for cloning Caf1a expression vectors, Stefan Wiemann (German Cancer Research Center, Heidelberg, Germany) for providing the human Pat1b cDNA clone, Ann-Bin Shyu (University of Texas, Houston, TX) for the Caf1a antibody, Mark Timmers (University of Utrecht) for Flag-Not1, Jens Lykke-Andersen (University of California at San Diego) for myc-Ccr4, Flag-Dcp1, and Flag-Dcp2, and Ulrike Engel (Nikon Imaging Center, University of Heidelberg) for generous support and advice on microscopy, as well as Witold Filipowicz (Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland) and Nancy Kedersha (Brigham and Woman's Hospital, Boston, MA) for helpful discussions on the manuscript.

This work was supported by young investigator grant HZ-NG-210 from the Helmholtz Gemeinschaft and research grant STO 859/2-1 from the Deutsche Forschungsgemeinschaft.