The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts.
Protein-RNA interactions are fundamental to core biological processes, such as mRNA splicing, localization, degradation, and translation. We developed a photoreactive nucleotide-enhanced UV crosslinking and oligo(dT) purification approach to identify the mRNA-bound proteome using quantitative proteomics and to display the protein occupancy on mRNA transcripts by next-generation sequencing. Application to a human embryonic kidney cell line identified close to 800 proteins. To our knowledge, nearly one-third were not previously annotated as RNA binding, and about 15% were not predictable by computational methods to interact with RNA. Protein occupancy profiling provides a transcriptome-wide catalog of potential cis-regulatory regions on mammalian mRNAs and showed that large stretches in 3' UTRs can be contacted by the mRNA-bound proteome, with numerous putative binding sites in regions harboring disease-associated nucleotide polymorphisms. Our observations indicate the presence of a large number of mRNA binders with diverse molecular functions participating in combinatorial posttranscriptional gene-expression networks.
Max Delbrück Center for Molecular Medicine, Berlin Institute for Medical Systems Biology, 13125 Berlin, Germany.
Alexander G
AG
Baltz
Mathias
M
Munschauer
Björn
B
Schwanhäusser
Alexandra
A
Vasile
Yasuhiro
Y
Murakawa
Markus
M
Schueler
Noah
N
Youngs
Duncan
D
Penfold-Brown
Kevin
K
Drew
Miha
M
Milek
Emanuel
E
Wyler
Richard
R
Bonneau
Matthias
M
Selbach
Christoph
C
Dieterich
Markus
M
Landthaler
D001665:Binding Sites; D002460:Cell Line; D006801:Humans; D013058:Mass Spectrometry; D040901:Proteomics / Q000379:methods*; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D017423:Sequence Analysis, RNA
D012333:RNA, Messenger; D016601:RNA-Binding Proteins