Roadblock termination by reb1p restricts cryptic and readthrough transcription.

Widely transcribed compact genomes must cope with the major challenge of frequent overlapping or concurrent transcription events. Efficient and timely transcription termination is crucial to control pervasive transcription and prevent transcriptional interference. In yeast, transcription termination of RNA polymerase II (RNAPII) occurs via two possible pathways that both require recognition of termination signals on nascent RNA by specific factors. We describe here an additional mechanism of transcription termination for RNAPII and demonstrate its biological significance. We show that the transcriptional activator Reb1p bound to DNA is a roadblock for RNAPII, which pauses and is ubiquitinated, thus triggering termination. Reb1p-dependent termination generates a class of cryptic transcripts that are degraded in the nucleus by the exosome. We also observed transcriptional interference between neighboring genes in the absence of Reb1p. This work demonstrates the importance of roadblock termination for controlling pervasive transcription and preventing transcription through gene regulatory regions.

Molecular basis for coordinating transcription termination with noncoding RNA degradation.

The Nrd1-Nab3-Sen1 (NNS) complex is essential for controlling pervasive transcription and generating sn/snoRNAs in S. cerevisiae. The NNS complex terminates transcription of noncoding RNA genes and promotes exosome-dependent processing/degradation of the released transcripts. The Trf4-Air2-Mtr4 (TRAMP) complex polyadenylates NNS target RNAs and favors their degradation. NNS-dependent termination and degradation are coupled, but the mechanism underlying this coupling remains enigmatic. Here we provide structural and functional evidence demonstrating that the same domain of Nrd1p interacts with RNA polymerase II and Trf4p in a mutually exclusive manner, thus defining two alternative forms of the NNS complex, one involved in termination and the other in degradation. We show that the Nrd1-Trf4 interaction is required for optimal exosome activity in vivo and for the stimulation of polyadenylation of NNS targets by TRAMP in vitro. We propose that transcription termination and RNA degradation are coordinated by switching between two alternative partners of the NNS complex.

The highly conserved eukaryotic DRG factors are required for efficient translation in a manner redundant with the putative RNA helicase Slh1.

Eukaryotic and archaeal DRG factors are highly conserved proteins with characteristic GTPase motifs. This suggests their implication in a central biological process, which has so far escaped detection. We show here that the two Saccharomyces cerevisiae DRGs form distinct complexes, RBG1 and RBG2, and that the former co-fractionate with translating ribosomes. A genetic screen for triple synthetic interaction demonstrates that yeast DRGs have redundant function with Slh1, a putative RNA helicase also associating with translating ribosomes. Translation and cell growth are severely impaired in a triple mutant lacking both yeast DRGs and Slh1, but not in double mutants. This new genetic assay allowed us to characterize the roles of conserved motifs present in these proteins for efficient translation and/or association with ribosomes. Altogether, our results demonstrate for the first time a direct role of the highly conserved DRG factors in translation and indicate that this function is redundantly shared by three factors. Furthermore, our data suggest that important cellular processes are highly buffered against external perturbation and, consequently, that redundantly acting factors may escape detection in current high-throughput binary genetic interaction screens.

Dissection of Dom34-Hbs1 reveals independent functions in two RNA quality control pathways.

Eukaryotic cells have several quality control pathways that rely on translation to detect and degrade defective RNAs. Dom34 and Hbs1 are two proteins that are related to translation termination factors and are involved in no-go decay (NGD) and nonfunctional 18S ribosomal RNA (rRNA) decay (18S NRD) pathways that eliminate RNAs that cause strong ribosomal stalls. Here we present the structure of Hbs1 with and without GDP and a low-resolution model of the Dom34-Hbs1 complex. This complex mimics complexes of the elongation factor and transfer RNA or of the translation termination factors eRF1 and eRF3, supporting the idea that it binds to the ribosomal A-site. We show that nucleotide binding by Hbs1 is essential for NGD and 18S NRD. Mutations in Hbs1 that disrupted the interaction between Dom34 and Hbs1 strongly impaired NGD but had almost no effect on 18S NRD. Hence, NGD and 18S NRD could be genetically uncoupled, suggesting that mRNA and rRNA in a stalled translation complex may not always be degraded simultaneously.

Futile cycle of transcription initiation and termination modulates the response to nucleotide shortage in S. cerevisiae.

Hidden transcription in eukaryotes carries a large potential of regulatory functions that are only recently beginning to emerge. Cryptic unstable transcripts (CUTs) are generated by RNA polymerase II (Pol II) and rapidly degraded after transcription in wild-type yeast cells. Whether CUTs or the act of transcription without RNA production have a function is presently unclear. We describe here a nonconventional mechanism of transcriptional regulation that relies on the selection of alternative transcription start sites to generate CUTs or mRNAs. Transcription from TATA box proximal start sites generates unstable transcripts and downregulates expression of the URA2 gene under repressing conditions. Uracil deprivation activates selection of distal start sites, leading to the production of stable mRNAs. We describe the elements that govern degradation of the CUT and activation of mRNA production by downstream transcription initiation. Importantly, we show that a similar mechanism applies to other genes in the nucleotides biogenesis pathway.

Mutations of RNA polymerase II activate key genes of the nucleoside triphosphate biosynthetic pathways.

The yeast URA2 gene, encoding the rate-limiting enzyme of UTP biosynthesis, is transcriptionally activated by UTP shortage. In contrast to other genes of the UTP pathway, this activation is not governed by the Ppr1 activator. Moreover, it is not due to an increased recruitment of RNA polymerase II at the URA2 promoter, but to its much more effective progression beyond the URA2 mRNA start site(s). Regulatory mutants constitutively expressing URA2 resulted from cis-acting deletions upstream of the transcription initiator region, or from amino-acid replacements altering the RNA polymerase II Switch 1 loop domain, such as rpb1-L1397S. These two mutation classes allowed RNA polymerase to progress downstream of the URA2 mRNA start site(s). rpb1-L1397S had similar effects on IMD2 (IMP dehydrogenase) and URA8 (CTP synthase), and thus specifically activated the rate-limiting steps of UTP, GTP and CTP biosynthesis. These data suggest that the Switch 1 loop of RNA polymerase II, located at the downstream end of the transcription bubble, may operate as a specific sensor of the nucleoside triphosphates available for transcription.

Deletion of EFL1 results in heterogeneity of the 60 S GTPase-associated rRNA conformation.

Previous work suggested that the release of the nucleolar Tif6 from nascent 60 S subunits occurs in the cytoplasm and requires the cytoplasmic EF-2-like GTPase, Efl1. To check whether this release involves an rRNA structural rearrangement mediated by Efl1, we analyzed the rRNA conformation of the GTPase center of 80 S ribosomes in three contexts: wild-type, Deltaefl1 and a dominant suppressor R1 of Deltaefl1. This analysis was restricted to domain II and VI of 25 S rRNA. The rRNA analysis of R1 ribosomes allows us to distinguish the effects due to depletion of Efl1 from the resulting nucleolar deficit of Tif6. Efl1 inhibits the EF-2 GTPase activity, suggesting that the two proteins share a similar ribosome-binding site. The 80 S ribosomes from either type failed to show any difference of conformation in the two rRNA domains analyzed. However, the same analysis performed on the pool of free 60 S subunits reveals several rRNA conformational differences between wild-type and Deltaefl1 subunits, whereas that from the suppressor strain is similar to wild-type. This suggests that the nucleolar deficit of Tif6 during assembly of the 60 S preribosomes is responsible for the changes in rRNA conformation observed in Deltaefl1 60 S subunits. We also purified 60 S preribosomes from the three genetic contexts by TAP-tagging Tif6. The protein content of 60 S preribosomes associated with Tif6p in a Deltaefl1 strain are obtained at a lower yield but have, surprisingly, a protein composition that is a priori similar to that of wild-type and the suppressor strain.

Mitochondrial DNA: an advance in eukaryotic cell biology in the 1960s.

Between 1950 and 1960 mitochondria were recognized as well-characterized organelles of animal and fungal cells. They shared more functional autonomy than other cellular structures. The transmission of some mitochondrial characteristics did not obey Mendelian rules and followed cytoplasmic inheritance patterns. Was this situation a consequence of still unknown complexities? We present a personal account on how approaches were set up to test very different hypotheses. In the end, it was shown that mitochondria had their own DNA, mitochondrial DNA, and that this molecule carried information specific to these organelles.

Pti1p and Ref2p found in association with the mRNA 3' end formation complex direct snoRNA maturation.

Eukaryotic RNA polymerase II transcribes precursors of mRNAs and of non-protein-coding RNAs such as snRNAs and snoRNAs. These RNAs have to be processed at their 3' ends to be functional. mRNAs are matured by cleavage and polyadenylation that require a well-characterized protein complex. Small RNAs are also subject to 3' end cleavage but are not polyadenylated. Here we show that two newly identified proteins, Pti1p and Ref2p, although they were found associated with the pre-mRNA 3' end processing complex, are essential for yeast snoRNA 3' end maturation. We also provide evidence that Pti1p probably acts by uncoupling cleavage and polyadenylation, and functions in coordination with the Nrd1p-dependent pathway for 3' end formation of non-polyadenylated transcripts.