The DEAD-box RNA helicase Dbp2p is highly conserved in eukaryotes and has been implicated in transcription, ribosome biogenesis, mRNP assembly, nuclear export, and long noncoding RNA (lncRNA) function. It is not understood how Dbp2p performs these seemingly unrelated biological roles. An important step toward addressing this question is the determination of cellular RNA binding sites of Dbp2p. Here, we identify transcriptome-wide RNA binding sites of Dbp2p from Saccharomyces cerevisiae using UV-crosslinking, denaturing tandem affinity purification, and next generation sequencing. We find that Dbp2p crosslinks to mRNAs and ribosomal RNAs, and markedly to noncoding RNAs, including snoRNA, snRNAs, and tRNAs. In snoRNAs, Dbp2p preferentially crosslinks at sites near the 3' ends. These sites coincide with regions where RNA-DNA hybrids (R-loops) form and with binding sites of Sen1p, another RNA helicase that functions in transcription termination and 3' processing of noncoding RNAs. We show that Dbp2p interacts in an RNA-independent manner with Sen1p in vivo. Dbp2p crosslinks to tRNAs and other RNAs also at sites where R-loops form. Collectively, our data link Dbp2p to noncoding RNAs, Sen1p, and R-loops. The transcriptome-wide connection to R-loops provides a unifying theme for diverse cellular roles of Dbp2p.
Keywords: NNS complex; R-loops; helicase; noncoding RNA; nucleus; snoRNA.
© 2018 Tedeschi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.