Transcriptome-wide analysis of alternative routes for RNA substrates into the exosome complex

The RNA exosome complex functions in both the accurate processing and rapid degradation of many different classes of RNA. Functional and structural analyses indicate that RNA can either be threaded through the central channel of the exosome or more directly access the active sites of the ribonucleases Rrp44 and Rrp6, but it was unclear how many substrates follow each pathway in vivo. To address this we used UV crosslinking in growing cells to identify transcriptome-wide interactions of RNAs with the major nuclear exosome-cofactor Mtr4 and with individual exosome subunits (Rrp6, Csl4, Rrp41 and Rrp44) along the threaded RNA path. We performed comparative analyses on exosome complexes lacking the exonucleolytic activity of Rrp44, either carrying a mutation in the Rrp44 S1 RNA-binding domain, predicted to disfavor direct access to the Rrp44 exonuclease active site, or with multiple mutations in Rrp41, reported to block RNA passage through the central channel. Our analysis identified targets using preferentially channel-threading pathway such as mRNAs, 5S rRNA or scR1. Our results suggest as well that aborted tRNAs transcripts, released during transcription, would be rapidly degraded using this route. Alternatively, pre-tRNAs appears to access Rrp44 directly. Both routes seems to be involved for degradation or maturation of RNAPI transcripts. The Rrp41 mutations were found to block substrate passage to Rrp44 only for cytoplasmic mRNAs, apparently confirming the prediction of widening of the lumen in the nuclear, Rrp6-associated complex. 13 samples were analysed from strains carrying HTP tagged protein. Duplicate CRAC experiments were carried out for each protein