The final step of cytoplasmic mRNA degradation proceeds in either a 5 0 -3 0 direction catalysed by Xrn1 or in a 3 0 -5 0 direction catalysed by the exosome. Dis3/Rrp44, an RNase II family protein, is the catalytic subunit of the exosome. In humans, there are three paralogues of this enzyme: DIS3, DIS3L, and DIS3L2. In this work, we identified a novel Schizosaccharomyces pombe exonuclease belonging to the conserved family of human DIS3L2 and plant SOV. Dis3L2 does not interact with the exosome components and localizes in the cytoplasm and in cytoplasmic foci, which are docked to P-bodies. Deletion of dis3l2 þ is synthetically lethal with xrn1D, while deletion of dis3l2 þ in an lsm1D background results in the accumulation of transcripts and slower mRNA degradation rates. Accumulated transcripts show enhanced uridylation and in vitro Dis3L2 displays a preference for uridylated substrates. Altogether, our results suggest that in S. pombe, and possibly in most other eukaryotes, Dis3L2 is an important factor in mRNA degradation. Therefore, this novel 3 0 -5 0 RNA decay pathway represents an alternative to degradation by Xrn1 and the exosome.

Telomeres protect the normal ends of chromosomes from being recognized as deleterious DNA double-strand breaks. Recent studies have uncovered an apparent paradox: although DNA repair is prevented, several proteins involved in DNA damage processing and checkpoint responses are recruited to telomeres in every cell cycle and are required for end protection1. It is currently not understood how telomeres prevent DNA damage responses from causing permanent cell cycle arrest. Here we show that fission yeast (Schizosaccharomyces pombe) cells lacking Taz1, an orthologue of human TRF1 and TRF2 (ref. 2), recruit DNA repair proteins (Rad22RAD52 and Rhp51RAD51, where the superscript indicates the human orthologue) and checkpoint sensors (RPA, Rad9, Rad26ATRIP and Cut5/Rad4TOPBP1) to telomeres. Despite this, telomeres fail to accumulate the checkpoint mediator Crb253BP1 and, consequently, do not activate Chk1-dependent cell cycle arrest. Artificially recruiting Crb253BP1 to taz1Δ telomeres results in a full checkpoint response and cell cycle arrest. Stable association of Crb253BP1 to DNA double-strand breaks requires two independent histone modifications: H4 dimethylation at lysine 20 (H4K20me2) and H2A carboxy-terminal phosphorylation (γH2A)3–5. Whereas γH2A can be readily detected, telomeres lack H4K20me2, in contrast to internal chromosome locations. Blocking checkpoint signal transduction at telomeres requires Pot1 and Ccq1, and loss of either Pot1 or Ccq1 from telomeres leads to Crb253BP1 foci formation, Chk1 activation and cell cycle arrest. Thus, telomeres constitute a chromatin-privileged region of the chromosomes that lack essential epigenetic markers for DNA damage response amplification and cell cycle arrest. Because the protein kinases ATM and ATR must associate with telomeres in each S phase to recruit telomerase6, exclusion of Crb253BP1 has a critical role in preventing telomeres from triggering cell cycle arrest.

Recent data reveal that a substantial fraction of transcripts generated by RNA polymerases I, II, and III are rapidly degraded in the nucleus by the combined action of the exosome and a noncanonical poly(A) polymerase activity. This work identifies a domain within the yeast nucleolus that is enriched in polyadenylated RNAs in the absence of the nuclear exosome RNase Rrp6 or the exosome cofactor Mtr4. In normal yeast cells, poly(A)؉ RNA was undetectable in the nucleolus but the depletion of either Rrp6 or Mtr4 led to the accumulation of polyadenylated RNAs in a discrete subnucleolar region. This nucleolar poly(A) domain is enriched for the U14 snoRNA and the snoRNP protein Nop1 but is distinct from the nucleolar body that functions in snoRNA maturation. In strains lacking both Rrp6 and the poly(A) polymerase Trf4, the accumulation of poly(A)؉ RNA was suppressed, suggesting the involvement of the Trf4-Air1/2-Mtr4 polyadenylation (TRAMP) complex. The accumulation of polyadenylated snoRNAs in a discrete nucleolar domain may promote their recognition as substrates for the exosome.A feature of RNAs in all organisms is that they are transcribed as precursors that undergo subsequent processing events to generate the mature functional forms. This was first found for the abundant cytoplasmic rRNAs, mRNAs, and tRNAs and was subsequently shown for the small nuclear RNAs (snRNAs), which participate in pre-mRNA splicing, and the small nucleolar RNAs (snoRNAs), which participate in rRNA processing and modification. Common steps in RNA maturation include endonuclease and exonuclease digestion, splicing, nucleotide modification, 3Ј poly(A) tail addition, and 5Ј capping. A major RNA processing activity is the multisubunit exosome complex (for reviews, see references 5, 11, and 34). The exosome contains multiple exoribonucleases that progressively digest RNAs from the 3Ј end. The exosome functions in both the accurate processing of some RNA species and the complete degradation of defective RNAs (35).The exosome was initially identified and characterized in the budding yeast Saccharomyces cerevisiae but is conserved in all eukaryotes analyzed, and a closely related complex is found in Archaea (20). Homologues of the yeast exosome proteins are present in humans (19), and at least four of them are functional when expressed in yeast, complementing mutations in the corresponding yeast genes (24). The yeast exosome is present in the nucleus, nucleolus, and cytoplasm and is comprised of 10 "core" subunits, all of which are essential for viability (reviewed in reference 11). An additional exonuclease, Rrp6, associates exclusively with the nuclear exosome in yeast (2, 4). The exosome complex purified from yeast lysates exhibited little activity (23), suggesting that its activation depends on additional factors that are not permanently associated with the complex. Consistent with this model, the exosome cofactor Mtr4 (a putative RNA helicase) was shown to associate with a poly(A) polymerase, Trf4, and one of two redundant zinc knuckle prote...

Small nucleolar RNAs (snoRNAs) associate with specific proteins forming small nucleolar ribonucleoprotein (snoRNP) particles, which are essential for ribosome biogenesis. The snoRNAs are transcribed, processed, and assembled in snoRNPs in the nucleoplasm. Mature particles are then transported to the nucleolus. In yeast, 3-end maturation of snoRNAs involves the activity of Rnt1p endonuclease and cleavage factor IA (CFIA). We report that after inhibition of CFIA components Rna14p and Rna15p, the snoRNP proteins Nop1p, Nop58p, and Gar1p delocalize from the nucleolus and accumulate in discrete nucleoplasmic foci. The U14 snoRNA, but not U3 snoRNA, similarly redistributes from the nucleolus to the nucleoplasmic foci. Simultaneous depletion of either Rna14p or Rna15p and the nuclear exosome component Rrp6p induces accumulation of poly(A)؉ RNA at the snoRNP-containing foci. We propose that the foci detected after CFIA inactivation correspond to quality control centers in the nucleoplasm. INTRODUCTIONSmall nucleolar RNAs (snoRNAs) are an abundant group of nonprotein coding RNAs in eukaryotic cells (Kiss, 2002). The snoRNAs associate with specific proteins forming small nucleolar ribonucleoprotein (snoRNP) particles, the majority of which participates in the biogenesis of ribosomes in the nucleolus (for recent reviews, see Bachellerie et al., 2002;Filipowicz and Pogacic, 2002;Kiss, 2002;Tran et al., 2004). The known snoRNAs fall into two major classes named box C/D and box H/ACA, which are characterized by distinctive sequence elements and associated proteins.The snoRNAs are transcribed as precursors that undergo posttranscriptional processing to generate the mature functional forms. Although most vertebrate snoRNAs are processed from the intronic regions of mRNA precursors Fournier, 1995, Bachellerie et al., 2002), only a few yeast snoRNAs are intron encoded. The majority of yeast snoRNAs are encoded in independent monocistronic or polycistronic transcripts that are transcribed by RNA polymerase II. Intronic snoRNAs can be produced either by splicing-dependent or by splicing-independent processing, whereas the maturation of independently transcribed units involves both endonucleolytic cleavage and exonucleolytic trimming (Filipowicz and Pogacic, 2002).In yeast, the U14 and U3 box C/D snoRNAs constitute paradigms for the study of snoRNA biosynthesis. U14 is cotranscribed with snR190, another box C/D snoRNA (Li et al., 1990). Cleavage of the dicistronic precursor by the endonuclease Rnt1 separates snR190 from U14 (Chanfreau et al., 1998). The pre-U3 transcript, by contrast, is produced from a single gene and contains the 7-methyl guanosine (m 7 G) cap structure, which is characteristic of mRNAs, and a short 3Ј extension. During maturation, the m 7 G cap on the pre-U3 is hypermethylated to 2,2,7-trimethylguanosine (TMG) and the 3Ј end is cleaved by Rnt1p, followed by removal of the 3Ј-terminal extension (Kufel et al., 2000). The yeast pre-U3 also contains an intron that is removed by the pre-mRNA splicing machinery, but t...

Big data (BD) analytics play a key role in helping hotel firms gain competitive advantages and achieve superior performance. The purpose of this study was to determine which factors encourage the use of big data analytics (BDA) by hotel firms and the impact of BDA on hotel firms’ performance. Understanding the impacts of big data analytics in the hotel sector is important to help hotel managers use big data for creating business value by increasing hotel performance. A research model was developed and tested with data collected through a questionnaire sent to hotel managers in a European country and analysed with PLS. The results indicate that organisational readiness and competitive pressure encourage the use of BDA through the mediating role of top management support. The findings also indicate that the use of BDA can create business value by increasing the main dimensions of hotel performance: financial performance, customer retention rate, and hotel reputation.

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