DNA-binding requirements of the yeast protein Rap1p as selected in silico from ribosomal protein gene promoter sequences.

Lascaris, Romeo; Mager, Willem H.; Planta, R.J.

doi:10.1093/bioinformatics/15.4.267

Cited by 91 publications

(82 citation statements)

References 38 publications

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“…Because the transcription rate of Rap1-target genes is extremely high during exponential growth of yeast, they might be highly susceptible to Kin28p inhibition by NA-PP1 treatment (17). As expected, we found in our array data that NA-PP1 treatment caused significant repression of ribosomal protein-encoding genes (Fig.…”

Section: Inhibition Of Kin28psupporting

confidence: 84%

Phosphorylation of the RNA polymerase II C-terminal domain by TFIIH kinase is not essential for transcription of Saccharomyces cerevisiae genome

Hong

Hong²,

Yoo

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Ser-5 phosphorylation of the RNA polymerase II (Pol II) C-terminal domain by TFIIH kinase has been implicated in critical steps in mRNA synthesis, such as Pol II promoter escape and mRNA 5 -capping. However, the general requirement and precise role of TFIIH kinase in Pol II transcription still remain elusive. Here we use a chemical genetics approach to show that, for a majority of budding-yeast genes, specific inhibition of the yeast TFIIH kinase results in a dramatic reduction in both mRNA level and Ser-5 C-terminal domain phosphorylation. Surprisingly, inhibition of TFIIH kinase activity only partially affected both Pol II density and Ser-2 phosphorylation level. The discrepancy between mRNA level and Pol II density is attributed to the defective 5 -capping, which results in the destabilization of mRNAs. Therefore, contrary to the current belief, our study points strongly toward a minor role of TFIIH kinase in Pol II transcription, and a more significant role in mRNA capping in budding yeast.T he C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) contains a series of YSPTSPS heptad repeats that are multiply-phosphorylated during the eukaryotic transcription cycle. Phosphorylation of the Ser-5 residue of the CTD heptad repeats has been implicated in multiple aspects of mRNA synthesis, such as promoter clearance (for transition from initiation to early elongation) and 5Ј-end capping of pre-mRNA (1). CTD Ser-2 phosphorylation has been implicated in productive elongation and the 3Ј-end processing of the transcript (2). However, it remains unclear whether CTD phosphorylation is required for transcription in general or functions in a promoter-and activatorspecific manner. Moreover, the requirement of Ser-5 phosphorylation for subsequent Ser-2 phosphorylation and transcriptional elongation remains controversial. An earlier study suggested that the CTD Ser-5 phosphorylation by Kin28 does not affect the Ser-2 phosphorylation level (3). In contrast, a recent study suggests that the CTD Ser-5 phosphorylation stimulates Ser-2 phosphorylation by BUR1/BUR2 kinase (4). These issues must be resolved to gain a clear understanding of the role of CTD phosphorylation in Pol II transcription in vivo.Temperature-sensitive mutants of CTD kinases have been used to study the functions of these enzymes in vivo (5). A genome-wide expression analysis using a temperature-sensitive mutant of KIN28, the Saccharomyces cerevisiae TFIIH kinase gene, showed that the loss of Kin28 function resulted in global shutdown of Pol II transcription (6). However, it has been demonstrated that the same mutation also disrupts other subunits in the TFIIH complex upon temperature shift, which makes the unambiguous functional analysis of this kinase difficult (7). Inhibition of CTD kinases with conventional pharmacological inhibitors is also problematic because these agents can nonspecifically inhibit other kinases (8). To overcome these obstacles, we used the ''analog-sensitive'' kinase-mutant strategy to dissect the unique roles of...

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Section: Inhibition Of Kin28psupporting

confidence: 84%

Phosphorylation of the RNA polymerase II C-terminal domain by TFIIH kinase is not essential for transcription of Saccharomyces cerevisiae genome

Hong

Hong²,

Yoo

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…Our analysis of yeast promoters encompassing Rap1p DNA-binding sites showed a strong bias for UASrpg-type sites at ribosomal gene promoters, where Rap1p probably behaves as a true activator. In about 50% of the cases, these genes show an arrangement of UASrpg sites similar to that of our pRPG2d plasmid (50). Note the good conservation of position T8, although the functional significance of this finding is unknown (6).…”

Section: Discussionsupporting

confidence: 66%

Alternative Mechanisms of Transcriptional Activation by Rap1p

Idrissi

García-Reyero

Fernández-Larrea

et al. 2001

Journal of Biological Chemistry

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“…Unexpectedly, 26 coding mRNAs appear in the 100 most Lhp1p-enriched RNAs with up to 300 in the top 500 (see Table 7, which is published as supporting information on the PNAS web site). Previous descriptions of La interactions with mRNAs have been restricted to virally encoded RNAs or endogenous mRNAs with IRES sequences or mRNAs with TOP sequences, which have not been documented in S. cerevisiae (32,33).…”

Section: Resultsmentioning

confidence: 99%

Identification of Lhp1p-associated RNAs by microarray analysis in Saccharomyces cerevisiae reveals association with coding and noncoding RNAs

Inada

Guthrie

2004

Proc. Natl. Acad. Sci. U.S.A.

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La is a conserved eukaryotic RNA-binding protein best known for its role in the biogenesis of noncoding RNAs transcribed by RNA polymerase III. To broaden our understanding of the function of the La homologous protein (Lhp1) in Saccharomyces cerevisiae, we have taken a genomics approach. Lhp1 ribonucleoprotein complexes were immunoprecipitated and bound RNAs were examined by hybridization to whole-genome microarrays that include >6,000 ORFs, documented noncoding RNAs, and the intervening intergenic regions. Demonstrating the validity of this approach, associations with previously known Lhp1p-associated RNAs were detected and associations with additional noncoding RNAs, including multiple tRNAs and small nucleolar RNAs, were revealed. Indicating that this approach provides a robust method for discovering RNAs, the data also identify associations between Lhp1p and several intergenic regions, three of which encode the recently annotated putative snoRNAs: RUF1, RUF2, and RUF3. Unexpectedly, we find that Lhp1p is also associated with a subset of coding mRNAs. These mRNAs include many ribosomal protein transcripts as well as the mRNA encoding Hac1p, a transcription factor required during the unfolded protein stress response. In cells lacking LHP1, Hac1p levels are decreased 2-to 3-fold, whereas no changes are detected in the levels of spliced or unspliced HAC1 mRNA or in the stability of Hac1p. Finally, although LHP1 is dispensable for growth under standard conditions, we find that it is required when the unfolded protein response is induced at elevated temperatures. These results suggest that Lhp1p may play a novel role in the translation of one or more cellular mRNAs. L a is an abundant eukaryotic RNA-binding protein implicated in multiple steps of RNA metabolism, including transcription and 3Ј end processing of RNA polymerase III (RNA pol III) transcripts, as well as translation of certain viral and endogenous mRNAs containing internal ribosome entry site (IRES) sequences (1). The best characterized role of La is in the biogenesis and processing of a variety of noncoding RNAs (1). La binds the 3Ј terminal UUU OH sequences of newly synthesized RNA pol III transcripts such as pre-tRNAs and pre-5S rRNA and protects them from degradation (2, 3). In yeast, La homologous protein (LHP1) is nonessential, which is surprising, given that its metazoan counterparts are involved in processing such a wide range of essential, noncoding RNAs. Nevertheless, genetic and biochemical analyses in Saccharomyces cerevisiae have confirmed that Lhp1p is involved in the processing of newly synthesized RNA pol III transcripts (4, 5), and revealed a similar role with noncoding RNAs generated by RNA pol II, spliceosomal small nuclear RNAs (snRNAs) (6), and U3 small nucleolar RNA (snoRNA) (7). Lhp1p is known to bind these noncoding RNA precursors and is thought to facilitate their maturation by stabilizing them from digestion. Because Lhp1p is not required for maintaining normal levels of mature ribonucleoproteins, it has been suggested that Lh...

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DNA-binding requirements of the yeast protein Rap1p as selected in silico from ribosomal protein gene promoter sequences.

Abstract: The rp database is available at the url: http://www. chem.vu.nl/BMB/Database.html.

Cited by 91 publications

References 38 publications

Phosphorylation of the RNA polymerase II C-terminal domain by TFIIH kinase is not essential for transcription of Saccharomyces cerevisiae genome

Phosphorylation of the RNA polymerase II C-terminal domain by TFIIH kinase is not essential for transcription of Saccharomyces cerevisiae genome

Alternative Mechanisms of Transcriptional Activation by Rap1p

Identification of Lhp1p-associated RNAs by microarray analysis in Saccharomyces cerevisiae reveals association with coding and noncoding RNAs

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