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1.  Molecular Basis for Coordinating Transcription Termination with Noncoding RNA Degradation 
Molecular Cell  2014;55(3):467-481.
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.
Graphical Abstract
•The Nrd1 CTD interaction domain (CID) recognizes a CTD mimic in Trf4•The CID interacts with RNAPII and Trf4 in a mutually exclusive manner•Architecture of the interactions between the NNS complex, the exosome, and TRAMP•The interaction of Nrd1 with Trf4 stimulates the polyadenylation activity of TRAMP
The Nrd1-Nab3-Sen1 complex controls pervasive transcription in yeast by terminating transcription of cryptic transcripts and directing these RNAs to degradation by the exosome. Tudek et al. show that the same domain of Nrd1p mediates mutually exclusive interactions with RNAPII or the exosome cofactor TRAMP to coordinate termination and RNA degradation.
PMCID: PMC4186968  PMID: 25066235
2.  Air2p is critical for the assembly and RNA-binding of the TRAMP complex and the KOW domain of Mtr4p is crucial for exosome activation 
Nucleic Acids Research  2012;40(12):5679-5693.
Trf4/5p-Air1/2p-Mtr4p polyadenylation complex (TRAMP) is an essential component of nuclear RNA surveillance in yeast. It recognizes a variety of nuclear transcripts produced by all three RNA polymerases, adds short poly(A) tails to aberrant or unstable RNAs and activates the exosome for their degradation. Despite the advances in understanding the structural features of the isolated complex subunits or their fragments, the details of complex assembly, RNA recognition and exosome activation remain poorly understood. Here we provide the first understanding of the RNA binding mode of the complex. We show that Air2p is an RNA-binding subunit of TRAMP. We identify the zinc knuckles (ZnK) 2, 3 and 4 as the RNA-binding domains, and reveal the essentiality of ZnK4 for TRAMP4 polyadenylation activity. Furthermore, we identify Air2p as the key component of TRAMP4 assembly providing bridging between Mtr4p and Trf4p. The former is bound via the N-terminus of Air2p, while the latter is bound via ZnK5, the linker between ZnK4 and 5 and the C-terminus of the protein. Finally, we uncover the RNA binding part of the Mtr4p arch, the KOW domain, as the essential component for TRAMP-mediated exosome activation.
PMCID: PMC3384339  PMID: 22402490
3.  Recognition of Transcription Termination Signal by the Nuclear Polyadenylated RNA-binding (NAB) 3 Protein* 
The Journal of Biological Chemistry  2010;286(5):3645-3657.
Non-coding RNA polymerase II transcripts are processed by the poly(A)-independent termination pathway that requires the Nrd1 complex. The Nrd1 complex includes two RNA-binding proteins, the nuclear polyadenylated RNA-binding (Nab) 3 and the nuclear pre-mRNA down-regulation (Nrd) 1 that bind their specific termination elements. Here we report the solution structure of the RNA-recognition motif (RRM) of Nab3 in complex with a UCUU oligonucleotide, representing the Nab3 termination element. The structure shows that the first three nucleotides of UCUU are accommodated on the β-sheet surface of Nab3 RRM, but reveals a sequence-specific recognition only for the central cytidine and uridine. The specific contacts we identified are important for binding affinity in vitro as well as for yeast viability. Furthermore, we show that both RNA-binding motifs of Nab3 and Nrd1 alone bind their termination elements with a weak affinity. Interestingly, when Nab3 and Nrd1 form a heterodimer, the affinity to RNA is significantly increased due to the cooperative binding. These findings are in accordance with the model of their function in the poly(A) independent termination, in which binding to the combined and/or repetitive termination elements elicits efficient termination.
PMCID: PMC3030368  PMID: 21084293
NMR; Protein Structure; RNA Processing; RNA-Protein Interaction; Transcription Termination
4.  1H, 13C, and 15N chemical shift assignments of ZCCHC9 
Biomolecular Nmr Assignments  2010;5(1):19-21.
ZCCHC9 is a human nuclear protein with sequence homology to yeast Air1p/Air2p proteins which are RNA-binding subunits of the Trf4/Air2/Mtr4 polyadenylation (TRAMP) complex involved in nuclear RNA quality control and degradation in yeast. The ZCCHC9 protein contains four retroviral-type zinc knuckle motifs. Here, we report the NMR spectral assignment of the zinc knuckle region of ZCCHC9. These data will allow performing NMR structural and RNA-binding studies of ZCCHC9 with the aim to investigate its role in the RNA quality control in human.
PMCID: PMC3049224  PMID: 20711761
ZCCHC9; Zinc knuckle; CCHC; RNA degradation
5.  Distinct Roles of Non-Canonical Poly(A) Polymerases in RNA Metabolism 
PLoS Genetics  2009;5(7):e1000555.
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.
Author Summary
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.
PMCID: PMC2700272  PMID: 19593367
6.  Draft Genome Sequence of the Sexually Transmitted Pathogen Trichomonas vaginalis 
Science (New York, N.Y.)  2007;315(5809):207-212.
We describe the genome sequence of the protist Trichomonas vaginalis, a sexually transmitted human pathogen. Repeats and transposable elements comprise about two-thirds of the ~160-megabase genome, reflecting a recent massive expansion of genetic material. This expansion, in conjunction with the shaping of metabolic pathways that likely transpired through lateral gene transfer from bacteria, and amplification of specific gene families implicated in pathogenesis and phagocytosis of host proteins may exemplify adaptations of the parasite during its transition to a urogenital environment. The genome sequence predicts previously unknown functions for the hydrogenosome, which support a common evolutionary origin of this unusual organelle with mitochondria.
PMCID: PMC2080659  PMID: 17218520

Results 1-6 (6)