SummaryThe 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.

SummaryGenomes are promiscuously transcribed, necessitating mechanisms that facilitate the sorting of RNA for function or destruction. The polyA (pA) tail is one such distinguishing feature, which in the Saccharomyces cerevisiae nucleus is bound by the Nab2p protein, yielding transcript protection. As Nab2p also contacts the main nuclear export factor Mex67p, we asked whether transport kinetics contributes to RNA sorting. Indeed, 3′ end sequencing of newly transcribed pA+ RNAs demonstrates that nuclear depletion of Mex67p elicits their instant and global decay. A similar phenotype is evident upon inactivation of other export factors and proportional to the amount of nuclear pA+ RNA. As RNA expression is partially rescued by Nab2p overexpression, we propose that an export block out-titrates Nab2p onto nuclear-retained pA+ RNA, reducing the pool of Nab2p available to protect new transcripts. More generally, we suggest that nuclear RNA decay, negotiated by Nab2p availability, aids in balancing cellular transcript supply with demand.

The polyadenosine tail (poly[A]-tail) is a universal modification of eukaryotic messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs). In budding yeast, Pap1-synthesized mRNA poly(A) tails enhance export and translation, whereas Trf4/5-mediated polyadenylation of ncRNAs facilitates degradation by the exosome. Using direct RNA sequencing, we decipher the extent of poly(A) tail dynamics in yeast defective in all relevant exonucleases, deadenylases, and poly(A) polymerases. Predominantly ncRNA poly(A) tails are 20-60 adenosines long. Poly(A) tails of newly transcribed mRNAs are 50 adenosine long on average, with an upper limit of 200. Exonucleolysis by Trf5-assisted nuclear exosome and cytoplasmic deadenylases trim the tails to 40 adenosines on average. Surprisingly, PAN2/3 and CCR4-NOT deadenylase complexes have a large pool of non-overlapping substrates mainly defined by expression level. Finally, we demonstrate that mRNA poly(A) tail length strongly responds to growth conditions, such as heat and nutrient deprivation.

Several trap plasmids (enabling positive selection of transposition events) were used to identify a pool of functional transposable elements (TEs) residing in bacteria of the genus Paracoccus (Alphaproteobacteria). Complex analysis of 25 strains representing 20 species of this genus led to the capture and characterization of (i) 37 insertion sequences (ISs) representing 9 IS families (IS3, IS5, IS6, IS21, IS66, IS256, IS1182, IS1380 and IS1634), (ii) a composite transposon Tn6097 generated by two copies of the ISPfe2 (IS1634 family) containing two predicted genetic modules, involved in the arginine deiminase pathway and daunorubicin/doxorubicin resistance, (iii) 3 non-composite transposons of the Tn3 family, including Tn5393 carrying streptomycin resistance and (iv) a transposable genomic island TnPpa1 (45 kb). Some of the elements (e.g. Tn5393, Tn6097 and ISs of the IS903 group of the IS5 family) were shown to contain strong promoters able to drive transcription of genes placed downstream of the target site of transposition. Through the application of trap plasmid pCM132TC, containing a promoterless tetracycline resistance reporter gene, we identified five ways in which transposition can supply promoters to transcriptionally silent genes. Besides highlighting the diversity and specific features of several TEs, the analyses performed in this study have provided novel and interesting information on (i) the dynamics of the process of transposition (e.g. the unusually high frequency of transposition of TnPpa1) and (ii) structural changes in DNA mediated by transposition (e.g. the generation of large deletions in the recipient molecule upon transposition of ISPve1 of the IS21 family). We also demonstrated the great potential of TEs and transposition in the generation of diverse phenotypes as well as in the natural amplification and dissemination of genetic information (of adaptative value) by horizontal gene transfer, which is considered the driving force of bacterial evolution.