Tho2-mediated escort of Nrd1 regulates the expression of aging-related genes.

The relationship between aging and RNA biogenesis and trafficking is attracting growing interest, yet the precise mechanisms are unknown. The THO complex is crucial for mRNA cotranscriptional maturation and export. Herein, we report that the THO complex is closely linked to the regulation of lifespan. Deficiencies in Hpr1 and Tho2, components of the THO complex, reduced replicative lifespan (RLS) and are linked to a novel Sir2-independent RLS control pathway. Although transcript sequestration in hpr1Δ or tho2Δ mutants was countered by exosome component Rrp6, loss of this failed to mitigate RLS defects in hpr1Δ. However, RLS impairment in hpr1Δ or tho2Δ was counteracted by the additional expression of Nrd1-specific mutants that interacted with Rrp6. This effect relied on the interaction of Nrd1, a transcriptional regulator of aging-related genes, including ribosome biogenesis or RNA metabolism genes, with RNA polymerase II. Nrd1 overexpression reduced RLS in a Tho2-dependent pathway. Intriguingly, Tho2 deletion mirrored Nrd1 overexpression effects by inducing arbitrary Nrd1 chromatin binding. Furthermore, our genome-wide ChIP-seq analysis revealed an increase in the recruitment of Nrd1 to translation-associated genes, known to be related to aging, upon Tho2 loss. Taken together, these findings underscore the importance of Tho2-mediated Nrd1 escorting in the regulation of lifespan pathway through transcriptional regulation of aging-related genes.

The Nrd1-Nab3-Sen1 transcription termination complex from a structural perspective.

A substantial part of living cells activity involves transcription regulation. The RNA polymerases responsible for this job need to know 'where/when' to start and stop in the genome, answers that may change throughout life and upon external stimuli. In Saccharomyces cerevisiae, RNA Pol II transcription termination can follow two different routes: the poly(A)-dependent one used for most of the mRNAs and the Nrd1/Nab3/Sen1 (NNS) pathway for non-coding RNAs (ncRNA). The NNS targets include snoRNAs and cryptic unstable transcripts (CUTs) generated by pervasive transcription. This review recapitulates the state of the art in structural biology and biophysics of the Nrd1, Nab3 and Sen1 components of the NNS complex, with special attention to their domain structures and interactions with peptide and RNA motifs, and their heterodimerization. This structural information is put into the context of the NNS termination mechanism together with possible prospects for evolution in the field.

Altered Levels of Negative Costimulatory Molecule V-Set Domain-Containing T-Cell Activation Inhibitor-1 (VTCN1) and Metalloprotease Nardilysin (NRD1) are Associated with Generalized Active Vitiligo.

BACKGROUND: Vitiligo is characterized by depigmented macules on the skin caused due to autoimmune destruction of melanocytes. V-set domain-containing T-cell activation inhibitor-1 (VTCN1) is a negative costimulatory molecule that plays a vital role in suppressing autoimmunity and tuning immune response. Nardilysin (NRD1), a metalloproteinase, cleaves membrane-tethered VTCN1 resulting in the shedding of soluble-VTCN1 (sVTCN1). However, the role of VTCN1 and NRD1 in vitiligo pathogenesis is unexplored. OBJECTIVES AND METHODS: This study was aimed to (i) Investigate the association of VTCN1 intronic polymorphisms (rs10923223 T/C and rs12046117 C/T) with vitiligo susceptibility in Gujarat population by using Polymerase Chain Reaction- Restriction Fragment Length Polymorphism (PCR-RFLP) (ii) Estimate VTCN1 & NRD1 transcript levels from peripheral blood mononuclear cells (PBMCs) and skin samples of vitiligo patients by real-time PCR, (iii) Estimate sVTCN1 and NRD1 protein levels from plasma by ELISA and (iv) Estimate VTCN1 protein levels in the skin samples of vitiligo patients by immunofluorescence. RESULTS: The analysis revealed increased VTCN1 and NRD1 transcript levels in the skin (p = .039, p = .021 respectively), increased sVTCN1 and NRD1 levels (p = .026, p = .015 respectively) in the plasma, and decreased VTCN1 protein levels (p = .0002) in the skin of vitiligo patients as compared to healthy controls. The genetic analysis revealed no significant association of VTCN1 intronic polymorphisms rs10923223 T/C and rs12046117 C/T with vitiligo susceptibility in Gujarat population (p = .359, p = .937, respectively). CONCLUSIONS: The present study revealed altered VTCN1 and NRD1 expressions in the blood and skin of vitiligo patients, suggesting their potential role in the development and progression of Vitiligo.

Data from crosslinking and analysis of cDNAs (CRAC) of Nab3 in yeast cells expressing a circular ncRNA decoy.

Pervasive transcription originating from the ubiquitous activity of RNA Polymerase II (RNAPII) generates a vast mass of non-coding RNAs (ncRNAs) that represent a potential harm to gene expression. In the compact genome of the yeast Saccharomyces cerevisiae, the main genomewide safeguard against pervasive ncRNAs is the Nrd1-Nab3-Sen1 (NNS) complex, composed of two RNA-binding proteins (Nrd1 and Nab3) and the helicase Sen1. The NNS complex directs transcription termination of ncRNA genes and promotes the rapid degradation of pervasive transcripts from yeast nuclei through its physical and functional coupling to the nuclear RNA exosome. We have recently shown that inhibition of the exosome in yeast cells leads to the accumulation of ncRNAs complexed with Nab3 and Nrd1, decreasing recycling of these termination factors to sites of transcription and inducing global termination defects at NNS targets. Consistent with the notion that ncRNAs out-titrate Nab3 and Nrd1 termination factors, we have shown that a similar genomewide termination impairment could be achieved by expressing a circular RNA decoy containing a Nab3 binding target [1]. In relation to this previous research article, here we expand our observations on the effect of the circular RNA decoy on NNS termination. We aimed at verifying that the Nab3 binding sequence present on the decoy is indeed efficiently sequestering Nab3 as intended by design, leading to the expected decrease of Nab3 binding on NNS targets. We employed the crosslinking and cDNA analysis protocol (CRAC) on yeast cells expressing the circular ncRNA decoy or a control construct. We present data from high-resolution genomewide RNA binding of Nab3 in three independent biological replicates of these S.cerevisiae cells, normalized by spiked-in S.pombe lysates. These data allow the useful assessment of the extent of co-transcriptional binding decrease of Nab3 by decoy ncRNA titration and will be valuable for further analyses of NNS targeting mechanisms.

Degradation of Non-coding RNAs Promotes Recycling of Termination Factors at Sites of Transcription.

A large share of the non-coding transcriptome in yeast is controlled by the Nrd1-Nab3-Sen1 (NNS) complex, which promotes transcription termination of non-coding RNA (ncRNA) genes, and by the nuclear exosome, which limits the steady-state levels of the transcripts produced. How unconstrained ncRNA levels affect RNA metabolism and gene expression are long-standing and important questions. Here, we show that degradation of ncRNAs by the exosome is required for freeing Nrd1 and Nab3 from the released transcript after termination. In exosome mutants, these factors are sequestered by ncRNAs and cannot be efficiently recycled to sites of transcription, inducing termination defects at NNS targets. ncRNA-dependent, genome-wide termination defects can be recapitulated by the expression of a degradation-resistant, circular RNA containing a natural NNS target in exosome-proficient cells. Our results have important implications for the mechanism of termination, the general impact of ncRNAs abundance, and the importance of nuclear ncRNA degradation.

Purification and In Vitro Analysis of the Exosome Cofactors Nrd1-Nab3 and Trf4-Air2.

In many eukaryotic organisms from yeast to human, the exosome plays an important role in the control of pervasive transcription and in non-coding RNA (ncRNA) processing and quality control by trimming precursor RNAs and degrading aberrant transcripts. In Saccharomyces cerevisiae this function is enabled by the interaction of the exosome with several cofactors: the Nrd1-Nab3 heterodimer and the Trf4-Air2-Mtr4 (TRAMP4) complex. Nrd1 and Nab3 are RNA binding proteins that recognize specific motifs enriched in the target ncRNAs, whereas TRAMP4 adds polyA tails at the 3' end of transcripts and stimulates RNA degradation by the exosome. This chapter provides protocols for the purification of recombinant forms of these exosome cofactors and for the in vitro assessment of their activity.

Domain selective labeling for NMR studies of multidomain proteins by domain ligation using highly active sortase A.

Structural study of multidomain proteins using NMR is an emerging issue for understanding biological functions. To this end, domain-specific labeling is expected to be a key technology for facilitating the NMR-assignment process and for collecting distance information via spin labeling. To obtain domain-specific labeled samples, use of sortase A as a protein ligation tool is a viable approach. Sortase A enables ligation of separately expressed proteins (domains) through the Leu-Pro-X-Thr-Gly linker. However, the ligation reaction mediated by sortase A is not efficient. Poor yield and long reaction times hamper large-scale preparation using sortase A. Here we report the application of highly active sortases to NMR analyses. Optimal yields can be achieved within several hours when the ligation reaction are mediated by highly active sortases at 4 °C. We propose that this protocol can contribute to structural analyses of multidomain proteins by NMR.

RNA Polymerase II CTD Tyrosine 1 Is Required for Efficient Termination by the Nrd1-Nab3-Sen1 Pathway.

In Saccharomyces cerevisiae, transcription termination at protein-coding genes is coupled to the cleavage of the nascent transcript, whereas most non-coding RNA transcription relies on a cleavage-independent termination pathway involving Nrd1, Nab3, and Sen1 (NNS). Termination involves RNA polymerase II CTD phosphorylation, but a systematic analysis of the contribution of individual residues would improve our understanding of the role of the CTD in this process. Here we investigated the effect of mutating phosphorylation sites in the CTD on termination. We observed widespread termination defects at protein-coding genes in mutants for Ser2 or Thr4 but rare defects in Tyr1 mutants for this genes class. Instead, mutating Tyr1 led to widespread termination defects at non-coding genes terminating via NNS. Finally, we showed that Tyr1 is important for pausing in the 5' end of genes and that slowing down transcription suppresses termination defects. Our work highlights the importance of Tyr1-mediated pausing in NNS-dependent termination.

RNA Polymerase II Transcription Attenuation at the Yeast DNA Repair Gene, DEF1, Involves Sen1-Dependent and Polyadenylation Site-Dependent Termination.

Termination of RNA Polymerase II (Pol II) activity serves a vital cellular role by separating ubiquitous transcription units and influencing RNA fate and function. In the yeast Saccharomyces cerevisiae, Pol II termination is carried out by cleavage and polyadenylation factor (CPF-CF) and Nrd1-Nab3-Sen1 (NNS) complexes, which operate primarily at mRNA and non-coding RNA genes, respectively. Premature Pol II termination (attenuation) contributes to gene regulation, but there is limited knowledge of its prevalence and biological significance. In particular, it is unclear how much crosstalk occurs between CPF-CF and NNS complexes and how Pol II attenuation is modulated during stress adaptation. In this study, we have identified an attenuator in the DEF1 DNA repair gene, which includes a portion of the 5'-untranslated region (UTR) and upstream open reading frame (ORF). Using a plasmid-based reporter gene system, we conducted a genetic screen of 14 termination mutants and their ability to confer Pol II read-through defects. The DEF1 attenuator behaved as a hybrid terminator, relying heavily on CPF-CF and Sen1 but without Nrd1 and Nab3 involvement. Our genetic selection identified 22 cis-acting point mutations that clustered into four regions, including a polyadenylation site efficiency element that genetically interacts with its cognate binding-protein Hrp1. Outside of the reporter gene context, a DEF1 attenuator mutant increased mRNA and protein expression, exacerbating the toxicity of a constitutively active Def1 protein. Overall, our data support a biologically significant role for transcription attenuation in regulating DEF1 expression, which can be modulated during the DNA damage response.

Cell-Cycle Modulation of Transcription Termination Factor Sen1.

Many non-coding transcripts (ncRNA) generated by RNA polymerase II in S. cerevisiae are terminated by the Nrd1-Nab3-Sen1 complex. However, Sen1 helicase levels are surprisingly low compared with Nrd1 and Nab3, raising questions regarding how ncRNA can be terminated in an efficient and timely manner. We show that Sen1 levels increase during the S and G2 phases of the cell cycle, leading to increased termination activity of NNS. Overexpression of Sen1 or failure to modulate its abundance by ubiquitin-proteasome-mediated degradation greatly decreases cell fitness. Sen1 toxicity is suppressed by mutations in other termination factors, and NET-seq analysis shows that its overexpression leads to a decrease in ncRNA production and altered mRNA termination. We conclude that Sen1 levels are carefully regulated to prevent aberrant termination. We suggest that ncRNA levels and coding gene transcription termination are modulated by Sen1 to fulfill critical cell cycle-specific functions.

A global function for transcription factors in assisting RNA polymerase II termination.

The role of transcription factors (TFs) on nucleosome positioning, RNA polymerase recruitment, and transcription initiation has been extensively characterized. Here, we propose that a subset of TFs such as Reb1, Abf1, Rap1, and TFIIIB also serve a major function in partitioning transcription units by assisting the Nrd1p-Nab3p-Sen1p Pol II termination pathway.

Chemical shift assignments of the first and second RRMs of Nrd1, a fission yeast MAPK-target RNA binding protein.

Negative regulator differentiation 1 (Nrd1), a fission yeast RNA binding protein, modulates cytokinesis and sexual development and contributes to stress granule formation in response to environmental stresses. Nrd1 comprises four RRM domains and binds and stabilizes Cdc4 mRNA that encodes the myosin II light chain. Nrd1 binds the Cpc2 fission-yeast RACK1 homolog, and the interaction promotes Nrd1 localization to stress granules. Interestingly, Pmk1 mitogen-activated protein kinase phosphorylates Thr40 in the unstructured N-terminal region and Thr126 in the first RRM domain of Nrd1. Phosphorylation significantly reduces RNA-binding activity and likely modulates Nrd1 function. To reveal the relationship between the structure and function of Nrd1 and how phosphorylation affects structure, we used heteronuclear NMR techniques to investigate the three-dimensional structure of Nrd1. Here we report the 1H, 13C, and 15N resonance assignments of RRM1-RRM2 (residues 108-284) comprising the first and second RRMs obtained using heteronuclear NMR techniques. Secondary structures derived from the chemical shifts are reported. These data should contribute to the understanding of the three-dimensional structure of the RRM1-RRM2 region of Nrd1 and the perturbation caused by phosphorylation.

Polyadenylation site selection: linking transcription and RNA processing via a conserved carboxy-terminal domain (CTD)-interacting protein.

Despite the fact that the process of mRNA polyadenylation has been known for more than 40 years, a detailed understating of the mechanism underlying polyadenylation site selection is still far from complete. As 3' end processing is intimately associated with RNA polymerase II (RNAPII) transcription, factors that can successively interact with the transcription machinery and recognize cis-acting sequences on the nascent pre-mRNA would be well suited to contribute to poly(A) site selection. Studies using the fission yeast Schizosaccharomyces pombe have recently identified Seb1, a protein that shares homology with Saccharomyces cerevisiae Nrd1 and human SCAF4/8, and that is critical for poly(A) site selection. Seb1 binds to the C-terminal domain (CTD) of RNAPII via a conserved CTD-interaction domain and recognizes specific sequence motifs clustered downstream of the polyadenylation site on the uncleaved pre-mRNA. In this short review, we summarize insights into Seb1-dependent poly(A) site selection and discuss some unanswered questions regarding its molecular mechanism and conservation.

Loss of Nardilysin, a Mitochondrial Co-chaperone for α-Ketoglutarate Dehydrogenase, Promotes mTORC1 Activation and Neurodegeneration.

We previously identified mutations in Nardilysin (dNrd1) in a forward genetic screen designed to isolate genes whose loss causes neurodegeneration in Drosophila photoreceptor neurons. Here we show that NRD1 is localized to mitochondria, where it recruits mitochondrial chaperones and assists in the folding of α-ketoglutarate dehydrogenase (OGDH), a rate-limiting enzyme in the Krebs cycle. Loss of Nrd1 or Ogdh leads to an increase in α-ketoglutarate, a substrate for OGDH, which in turn leads to mTORC1 activation and a subsequent reduction in autophagy. Inhibition of mTOR activity by rapamycin or partially restoring autophagy delays neurodegeneration in dNrd1 mutant flies. In summary, this study reveals a novel role for NRD1 as a mitochondrial co-chaperone for OGDH and provides a mechanistic link between mitochondrial metabolic dysfunction, mTORC1 signaling, and impaired autophagy in neurodegeneration.

The Nrd1-like protein Seb1 coordinates cotranscriptional 3' end processing and polyadenylation site selection.

Termination of RNA polymerase II (RNAPII) transcription is associated with RNA 3' end formation. For coding genes, termination is initiated by the cleavage/polyadenylation machinery. In contrast, a majority of noncoding transcription events in Saccharomyces cerevisiae does not rely on RNA cleavage for termination but instead terminates via a pathway that requires the Nrd1-Nab3-Sen1 (NNS) complex. Here we show that the Schizosaccharomyces pombe ortholog of Nrd1, Seb1, does not function in NNS-like termination but promotes polyadenylation site selection of coding and noncoding genes. We found that Seb1 associates with 3' end processing factors, is enriched at the 3' end of genes, and binds RNA motifs downstream from cleavage sites. Importantly, a deficiency in Seb1 resulted in widespread changes in 3' untranslated region (UTR) length as a consequence of increased alternative polyadenylation. Given that Seb1 levels affected the recruitment of conserved 3' end processing factors, our findings indicate that the conserved RNA-binding protein Seb1 cotranscriptionally controls alternative polyadenylation.

Fail-safe transcription termination: Because one is never enough.

Termination of RNA polymerase II (RNAPII) transcription is a fundamental step of gene expression that involves the release of the nascent transcript and dissociation of RNAPII from the DNA template. As transcription termination is intimately linked to RNA 3' end processing, termination pathways have a key decisive influence on the fate of the transcribed RNA. Quite remarkably, when reaching the 3' end of genes, a substantial fraction of RNAPII fail to terminate transcription, requiring the contribution of alternative or "fail-safe" mechanisms of termination to release the polymerase. This point of view covers redundant mechanisms of transcription termination and how they relate to conventional termination models. In particular, we expand on recent findings that propose a reverse torpedo model of termination, in which the 3'5' exonucleolytic activity of the RNA exosome targets transcription events associated with paused and backtracked RNAPII.

Termination of Transcription of Short Noncoding RNAs by RNA Polymerase II.

The RNA polymerase II transcription cycle is often divided into three major stages: initiation, elongation, and termination. Research over the last decade has blurred these divisions and emphasized the tightly regulated transitions that occur as RNA polymerase II synthesizes a transcript from start to finish. Transcription termination, the process that marks the end of transcription elongation, is regulated by proteins that interact with the polymerase, nascent transcript, and/or chromatin template. The failure to terminate transcription can cause accumulation of aberrant transcripts and interfere with transcription at downstream genes. Here, we review the mechanism, regulation, and physiological impact of a termination pathway that targets small noncoding transcripts produced by RNA polymerase II. We emphasize the Nrd1-Nab3-Sen1 pathway in yeast, in which the process has been extensively studied. The importance of understanding small RNA termination pathways is underscored by the need to control noncoding transcription in eukaryotic genomes.

NRD1, which encodes nardilysin protein, promotes esophageal cancer cell invasion through induction of MMP2 and MMP3 expression.

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide. In the present study, to identify novel prognostic markers or therapeutic targets for ESCC, we reviewed a list of genes with upregulated expression in ESCC compared with normal esophagus, as identified by our serial analysis of gene expression (SAGE) analysis. We focused on the NRD1 gene, which encodes the nardilysin protein. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in 34 ESCC tissue samples revealed that mRNA expression of NRD1 was upregulated in 56% of ESCC tissue samples. Immunohistochemical analysis of nardilysin in 109 ESCC tissue samples demonstrated that 43 (39%) ESCC cases were positive for nardilysin. Nardilysin-positive ESCC cases were more advanced in terms of T classification (P = 0.0007), N classification (P = 0.0164), and tumor stage (P < 0.0001) than nardilysin-negative ESCC cases. Furthermore, nardilysin expression was significantly associated with poorer prognosis (P = 0.0258). Univariate and multivariate analyses revealed that nardilysin expression is an independent prognostic classifier of patients with ESCC. The invasiveness of NRD1-knockdown TE1 and TE5 esophageal cancer cell lines was less than that of the negative control siRNA-transfected cell lines. Expression of MMP2 and MMP3 mRNA was significantly lower in NRD1-knockdown TE5 cells than in negative control siRNA-transfected cells. These results suggest that nardilysin is involved in tumor progression, and is an independent prognostic classifier in patients with ESCC.

The RNA polymerase II C-terminal domain-interacting domain of yeast Nrd1 contributes to the choice of termination pathway and couples to RNA processing by the nuclear exosome.

The RNA polymerase II (RNApII) C-terminal domain (CTD)-interacting domain (CID) proteins are involved in two distinct RNApII termination pathways and recognize different phosphorylated forms of CTD. To investigate the role of differential CTD-CID interactions in the choice of termination pathway, we altered the CTD-binding specificity of Nrd1 by domain swapping. Nrd1 with the CID from Rtt103 (Nrd1(CID(Rtt103))) causes read-through transcription at many genes, but can also trigger termination where multiple Nrd1/Nab3-binding sites and the Ser(P)-2 CTD co-exist. Therefore, CTD-CID interactions target specific termination complexes to help choose an RNApII termination pathway. Interactions of Nrd1 with both CTD and nascent transcripts contribute to efficient termination by the Nrd1 complex. Surprisingly, replacing the Nrd1 CID with that from Rtt103 reduces binding to Rrp6/Trf4, and RNA transcripts terminated by Nrd1(CID(Rtt103)) are predominantly processed by core exosome. Thus, the Nrd1 CID couples Ser(P)-5 CTD not only to termination, but also to RNA processing by the nuclear exosome.

A network of interdependent molecular interactions describes a higher order Nrd1-Nab3 complex involved in yeast transcription termination.

Nab3 and Nrd1 are yeast heterogeneous nuclear ribonucleoprotein (hnRNP)-like proteins that heterodimerize and bind RNA. Genetic and biochemical evidence reveals that they are integral to the termination of transcription of short non-coding RNAs by RNA polymerase II. Here we define a Nab3 mutation (nab3Δ134) that removes an essential part of the protein's C terminus but nevertheless can rescue, in trans, the phenotype resulting from a mutation in the RNA recognition motif of Nab3. This low complexity region of Nab3 appears intrinsically unstructured and can form a hydrogel in vitro. These data support a model in which multiple Nrd1-Nab3 heterodimers polymerize onto substrate RNA to effect termination, allowing complementation of one mutant Nab3 molecule by another lacking a different function. The self-association property of Nab3 adds to the previously documented interactions between these hnRNP-like proteins, RNA polymerase II, and the nascent transcript, leading to a network of nucleoprotein interactions that define a higher order Nrd1-Nab3 complex. This was underscored from the synthetic phenotypes of yeast strains with pairwise combinations of Nrd1 and Nab3 mutations known to affect their distinct biochemical activities. The mutations included a Nab3 self-association defect, a Nab3-Nrd1 heterodimerization defect, a Nrd1-polymerase II binding defect, and an Nab3-RNA recognition motif mutation. Although no single mutation was lethal, cells with any two mutations were not viable for four such pairings, and a fifth displayed a synthetic growth defect. These data strengthen the idea that a multiplicity of interactions is needed to assemble a higher order Nrd1-Nab3 complex that coats specific nascent RNAs in preparation for termination.