Rio1 downregulates centromeric RNA levels to promote the timely assembly of structurally fit kinetochores.

Kinetochores assemble on centromeres (CENs) via histone H3 variant CENP-A and low levels of CEN transcripts. RNA polymerase II (RNAPII) activity is restrained by the CEN histone code, while CEN RNA concentrations are reduced by the nuclear exosome. Using S. cerevisiae, we add kinase Rio1 to this scheme as it downregulates RNAPII, and promotes CEN RNA turnover via exoribonuclease Rat1. Transcription factor Cbf1 and the assembled kinetochore further restrain CEN transcription. CEN transcripts exist as long (up to 11,000nt) and short RNAs (119±40nt), which may underlie CEN identity and kinetochore recruitment. While also curtailed by Rio1, Rat1, and the exosome, periCEN RNAs (<200nt) accumulate at levels that are one order of magnitude higher than the CEN transcripts. Depleting Rio1 causes CEN and periCEN RNA buildup, kinetochore malformation, and chromosome loss. Depleting human orthologue RioK1 leads to CEN RNA accumulation and micronuclei formation, suggesting that Rio1/RioK1 activity at centromeres is conserved.

Unraveling the stepwise maturation of the yeast telomerase including a Cse1 and Mtr10 mediated quality control checkpoint

Telomerases elongate the ends of chromosomes required for cell immortality through their reverse transcriptase activity. By using the model organism Saccharomyces cerevisiae we defined the order in which the holoenzyme matures. First, a longer precursor of the telomerase RNA, TLC1 is transcribed and exported into the cytoplasm, where it associates with the protecting Sm-ring, the Est and the Pop proteins. This partly matured telomerase is re-imported into the nucleus via Mtr10 and a novel TLC1-import factor, the karyopherin Cse1. Remarkably, while mutations in all known transport factors result in short telomere ends, mutation in CSE1 leads to the amplification of Y′ elements in the terminal chromosome regions and thus elongated telomere ends. Cse1 does not only support TLC1 import, but also the Sm-ring stabilization on the RNA enableling Mtr10 contact and nuclear import. Thus, Sm-ring formation and import factor contact resembles a quality control step in the maturation process of the telomerase. The re-imported immature TLC1 is finally trimmed into the 1158 nucleotides long mature form via the nuclear exosome. TMG-capping of TLC1 finalizes maturation, leading to mature telomerase.

Global view on the metabolism of RNA poly(A) tails in yeast Saccharomyces cerevisiae

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.

The leucine-NH4+ uptake regulator Any1 limits growth as part of a general amino acid control response to loss of La protein by fission yeast

The sla1+ gene of Schizosachharoymces pombe encodes La protein which promotes proper processing of precursor-tRNAs. Deletion of sla1 (sla1Δ) leads to disrupted tRNA processing and sensitivity to target of rapamycin (TOR) inhibition. Consistent with this, media containing NH4+ inhibits leucine uptake and growth of sla1Δ cells. Here, transcriptome analysis reveals that genes upregulated in sla1Δ cells exhibit highly significant overalp with general amino acid control (GAAC) genes in relevant transcriptomes from other studies. Growth in NH4+ media leads to additional induced genes that are part of a core environmental stress response (CESR). The sla1Δ GAAC response adds to evidence linking tRNA homeostasis and broad signaling in S. pombe. We provide evidence that deletion of the Rrp6 subunit of the nuclear exosome selectively dampens a subset of GAAC genes in sla1Δ cells suggesting that nuclear surveillance-mediated signaling occurs in S. pombe. To study the NH4+-effects, we isolated sla1Δ spontaneous revertants (SSR) of the slow growth phenotype and found that GAAC gene expression and rapamycin hypersensitivity were also reversed. Genome sequencing identified a F32V substitution in Any1, a known negative regulator of NH4+-sensitive leucine uptake linked to TOR. We show that 3H-leucine uptake by SSR-any1-F32V cells in NH4+-media is more robust than by sla1Δ cells. Moreover, F32V may alter any1+ function in sla1Δ vs. sla1+ cells in a distinctive way. Thus deletion of La, a tRNA processing factor leads to a GAAC response involving reprogramming of amino acid metabolism, and isolation of the any1-F32V rescuing mutant provides an additional specific link.

The zinc-finger protein Red1 orchestrates MTREC submodules and binds the Mtl1 helicase arch domain

Cryptic unstable transcripts (CUTs) are rapidly degraded by the nuclear exosome in a process requiring the RNA helicase Mtr4 and specific adaptor complexes for RNA substrate recognition. The PAXT and MTREC complexes have recently been identified as homologous exosome adaptors in human and fission yeast, respectively. The eleven-subunit MTREC comprises the zinc-finger protein Red1 and the Mtr4 homologue Mtl1. Here, we use yeast two-hybrid and pull-down assays to derive a detailed interaction map. We show that Red1 bridges MTREC submodules and serves as the central scaffold. In the crystal structure of a minimal Mtl1/Red1 complex an unstructured region adjacent to the Red1 zinc-finger domain binds to both the Mtl1 KOW domain and stalk helices. This interaction extends the canonical interface seen in Mtr4-adaptor complexes. In vivo mutational analysis shows that this interface is essential for cell survival. Our results add to Mtr4 versatility and provide mechanistic insights into the MTREC complex.

Nuclear and cytoplasmic RNA exosomes and PELOTA1 prevent miRNA-induced secondary siRNA production in Arabidopsis

Amplification of short interfering RNA (siRNAs) via RNA dependent RNA Polymerases (RdRPs) is of fundamental importance in RNA silencing. In plants, silencing by microRNAs (miRNAs) generally does not lead to engagement of RdRPs, in part thanks to an as yet poorly understood activity of the cytoplasmic exosome adaptor SKI2. Here, we show that mutation of the cytoplasmic exosome subunit RRP45B results in siRNA production very similar to what is observed in ski2 mutants. Furthermore, loss of the nuclear exosome adaptor HEN2 leads to secondary siRNA production from miRNA targets largely distinct from those producing siRNAs in ski2. Importantly, mutation of the Release Factor paralogue PELOTA1 required for subunit dissociation of stalled ribosomes causes siRNA production from miRNA targets overlapping with, but distinct from, those affected in ski2 and rrp45b mutants. We also show that miRNA-induced illicit secondary siRNA production correlates with miRNA levels rather than accumulation of stable 5'-cleavage fragments. We propose that stalled RNA-induced Silencing Complex (RISC) and ribosomes, but not stable target mRNA cleavage fragments released from RISC, trigger secondary siRNA production, and that the exosome limits siRNA amplification by reducing RISC dwell time on miRNA target mRNAs while PELOTA1 does so by reducing ribosome stalling.

The inner nuclear membrane protein Lem2 coordinates RNA degradation at the nuclear periphery

Transcriptionally silent chromatin often localizes to the nuclear periphery. However, whether the nuclear envelope (NE) is a site for post-transcriptional gene repression is unknown. Here we demonstrate that S. pombe Lem2, an NE protein, regulates nuclear exosome-mediated RNA degradation. Lem2 deletion causes accumulation of non-coding RNAs and meiotic transcripts. Indeed, an engineered exosome substrate RNA shows Lem2-dependent localization to the nuclear periphery. Lem2 does not directly bind RNA, but instead physically interacts with the exosome-targeting MTREC complex and promotes RNA recruitment. The Lem2-assisted pathway acts independently of nuclear bodies where exosome factors assemble, revealing that multiple spatially distinct degradation pathways exist. The Lem2 pathway is environmentally responsive: nutrient availability modulates Lem2 regulation of meiotic transcripts. Our data indicate that Lem2 recruits exosome co-factors to the nuclear periphery to coordinate RNA surveillance and regulates transcripts during the mitosis-to-meiosis switch.

The CTEXT complex in Saccharomyces cerevisiae plays a crucial role in degrading distinct sets of aberrant mRNAs by the nuclear exosome

In Saccharomyces cerevisiae, DRN (Decay of RNA in the Nucleus) requiring Cbc1/2p, Tif4631p, and Upf3p promotes the exosomal degradation of aberrantly long 3′-extended-, export-defective transcripts and a small group of normal (special) mRNAs. In this study, using a systematic proteomic analysis we show that each of the known components interacts with one another and they exist as a separate complex, which was dubbed CTEXT (CBC-Tif4631p-dependent EXosome Targeting). We also identified a DEAD-box RNA helicase Dbp2p as an additional novel component of CTEXT during this analysis which was further bolstered by the finding that genomic deletions of Dbp2p led to the stabilization of all the signature nuclear messages. Interestingly, the RRM domain of Tif4631p located at the extreme N-termini of this polypeptide was found to play a vital role in in mediating the interaction of the CTEXT with the core exosome complex. These inferences were substantiated by the finding that deletion of this domain led to the functional impairment of the CTEXT complex. Thus, the CTEXT constitutes an independent complex that assists the nuclear exosome in degrading the select classes of nuclear transcripts in Saccharomyces cerevisiae.

ZCCHC8 is required for the degradation of pervasive transcripts originating from multiple genomic regulatory features

ABSTRACTThe vast majority of mammalian genomes are transcribed as non-coding RNA in what is referred to as “pervasive transcription.” Recent studies have uncovered various families of non-coding RNA transcribed upstream of transcription start sites. In particular, highly unstable promoter upstream transcripts known as PROMPTs have been shown to be targeted for exosomal degradation by the nuclear exosome targeting complex (NEXT) consisting of the RNA helicase MTR4, the zinc-knuckle scaffold ZCCHC8, and the RNA binding protein RBM7. Here, we report that in addition to its known RNA substrates, ZCCHC8 is required for the targeted degradation of pervasive transcripts produced at CTCF binding sites, open chromatin regions, promoters, promoter flanking regions, and transcription factor binding sites. Additionally, we report that a significant number of RIKEN cDNAs and predicted genes display the hallmarks of PROMPTs and are also substrates for ZCCHC8 and/or NEXT complex regulation suggesting these are unlikely to be functional genes. Our results suggest that ZCCHC8 and/or the NEXT complex may play a larger role in the global regulation of pervasive transcription than previously reported.