The PUF binding landscape in metazoan germ cells

PUF (Pumilio/FBF) proteins are RNA-binding proteins and conserved stem cell regulators. The Caenorhabditis elegans PUF proteins FBF-1 and FBF-2 (collectively FBF) regulate mRNAs in germ cells. Without FBF, adult germlines lose all stem cells. A major gap in our understanding of PUF proteins, includi...

Full description

Saved in:
Bibliographic Details
Published in:RNA (Cambridge) 2016-07, Vol.22 (7), p.1026-1043
Main Authors: Prasad, Aman, Porter, Douglas F, Kroll-Conner, Peggy L, Mohanty, Ipsita, Ryan, Anne R, Crittenden, Sarah L, Wickens, Marvin, Kimble, Judith
Format: Article
Language:English
Subjects:
Animals
Caenorhabditis elegans
Caenorhabditis elegans Proteins - genetics
Caenorhabditis elegans Proteins - metabolism
Germ Cells - metabolism
Metazoa
Protein Binding
RNA, Messenger - metabolism
RNA-Binding Proteins - genetics
RNA-Binding Proteins - metabolism
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:PUF (Pumilio/FBF) proteins are RNA-binding proteins and conserved stem cell regulators. The Caenorhabditis elegans PUF proteins FBF-1 and FBF-2 (collectively FBF) regulate mRNAs in germ cells. Without FBF, adult germlines lose all stem cells. A major gap in our understanding of PUF proteins, including FBF, is a global view of their binding sites in their native context (i.e., their "binding landscape"). To understand the interactions underlying FBF function, we used iCLIP (individual-nucleotide resolution UV crosslinking and immunoprecipitation) to determine binding landscapes of C. elegans FBF-1 and FBF-2 in the germline tissue of intact animals. Multiple iCLIP peak-calling methods were compared to maximize identification of both established FBF binding sites and positive control target mRNAs in our iCLIP data. We discovered that FBF-1 and FBF-2 bind to RNAs through canonical as well as alternate motifs. We also analyzed crosslinking-induced mutations to map binding sites precisely and to identify key nucleotides that may be critical for FBF-RNA interactions. FBF-1 and FBF-2 can bind sites in the 5'UTR, coding region, or 3'UTR, but have a strong bias for the 3' end of transcripts. FBF-1 and FBF-2 have strongly overlapping target profiles, including mRNAs and noncoding RNAs. From a statistically robust list of 1404 common FBF targets, 847 were previously unknown, 154 were related to cell cycle regulation, three were lincRNAs, and 335 were shared with the human PUF protein PUM2.
ISSN:1355-8382
1469-9001
DOI:10.1261/rna.055871.116