Trf4p and Trf5p are non-canonical poly(A) polymerases and are part of the heteromeric protein complexes TRAMP4 and TRAMP5 that promote the degradation of aberrant and short-lived RNA substrates by interacting with the nuclear exosome. To assess the level of functional redundancy between the paralogous Trf4 and Trf5 proteins and to investigate the role of the Trf4-dependent polyadenylation in vivo, we used DNA microarrays to compare gene expression of the wild-type yeast strain of S. cerevisiae with either that of trf4Δ or trf5Δ mutant strains or the trf4Δ mutant expressing the polyadenylation-defective Trf4(DADA) protein. We found little overlap between the sets of transcripts with altered expression in the trf4Δ or the trf5Δ mutants, suggesting that Trf4p and Trf5p target distinct groups of RNAs for degradation. Surprisingly, most RNAs the expression of which was altered by the trf4 deletion were restored to wild-type levels by overexpression of TRF4(DADA), showing that the polyadenylation activity of Trf4p is dispensable in vivo. Apart from previously reported Trf4p and Trf5p target RNAs, this analysis along with in vivo cross-linking and RNA immunopurification-chip experiments revealed that both the TRAMP4 and the TRAMP5 complexes stimulate the degradation of spliced-out introns via a mechanism that is independent of the polyadenylation activity of Trf4p. In addition, we show that disruption of trf4 causes severe shortening of telomeres suggesting that TRF4 functions in the maintenance of telomere length. Finally, our study demonstrates that TRF4, the exosome, and TRF5 participate in antisense RNA–mediated regulation of genes involved in phosphate metabolism. In conclusion, our results suggest that paralogous TRAMP complexes have distinct RNA selectivities with functional implications in RNA surveillance as well as other RNA–related processes. This indicates widespread and integrative functions of TRAMP complexes for the coordination of different gene expression regulatory processes.
The discovery that most regions of the genome are actively transcribed into non-coding RNAs has dramatically increased interest in their function and regulation. Recent data from us and others have shed light on the molecular machinery that promotes the decay of such transcripts. In the yeast S. cerevisiae, Trf4p and Trf5p are alternative subunits of the so-called TRAMP complex, which degrades aberrant and short-lived RNAs. They add short poly(A) tails to their substrate RNAs that function as landing pads for exonucleases mediating RNA decay. Although alternate compositions of TRAMP complexes exist, the RNA substrate specificities and the processes controlled by them have not been determined. Applying a genome-wide approach, we describe overlapping yet distinct functional implications of different TRAMP complexes, and we demonstrate strong connections between RNA quality control and other RNA–related processes such as telomer length maintenance. Moreover, our study shows that the degradation of specific target RNAs is not strictly dependent on the polyadenylation activity of Trf proteins in vivo. These results suggest novel and integrative functions of TRAMP complexes for RNA regulation.