Damien Hermand

The conserved Wobble uridine tRNA thiolase Ctu1-Ctu2 is required to maintain genome integrity.

Modified nucleosides close to the anticodon are important for the proper decoding of mRNA by the ribosome. Particularly, the uridine at the first anticodon position (U34) of glutamate, lysine, and glutamine tRNAs is universally thiolated (S2U34), which is proposed to be crucial for both restriction of wobble in the corresponding split codon box and efficient codon–anticodon interaction. Here we show that the highly conserved complex Ctu1–Ctu2 (cytosolic thiouridylase) is responsible for the 2-thiolation of cytosolic tRNAs in the nematode and fission yeast. In both species, inactivation of the complex leads to loss of thiolation on tRNAs and to a thermosensitive decrease of viability associated with marked ploidy abnormalities and aberrant development. Increased level of the corresponding tRNAs suppresses the fission yeast defects, and our data suggest that these defects could result from both misreading and frame shifting during translation. Thus, a translation defect due to unmodified tRNAs results in severe genome instability.

Modification of tRNALysUUU by Elongator Is Essential for Efficient Translation of Stress mRNAs

The Elongator complex, including the histone acetyl transferase Sin3/Elp3, was isolated as an RNA polymerase II-interacting complex, and cells deficient in Elongator subunits display transcriptional defects. However, it has also been shown that Elongator mediates the modification of some tRNAs, modulating translation efficiency. We show here that the fission yeast Sin3/Elp3 is important for oxidative stress survival. The stress transcriptional program, governed by the Sty1-Atf1-Pcr1 pathway, is affected in mutant cells, but not severely. On the contrary, cells lacking Sin3/Elp3 cannot modify the uridine wobble nucleoside of certain tRNAs, and other tRNA modifying activities such as Ctu1-Ctu2 are also essential for normal tolerance to H2O2. In particular, a plasmid over-expressing the tRNALys UUU complements the stress-related phenotypes of Sin3/Elp3 mutant cells. We have determined that the main H2O2-dependent genes, including those coding for the transcription factors Atf1 and Pcr1, are highly expressed mRNAs containing a biased number of lysine-coding codons AAA versus AAG. Thus, their mRNAs are poorly translated after stress in cells lacking Sin3/Elp3 or Ctu2, whereas a mutated atf1 transcript with AAA-to-AAG lysine codons is efficiently translated in all strain backgrounds. Our study demonstrates that the lack of a functional Elongator complex results in stress phenotypes due to its contribution to tRNA modification and subsequent translation inefficiency of certain stress-induced, highly expressed mRNAs. These results suggest that the transcriptional defects of these strain backgrounds may be a secondary consequence of the deficient expression of a transcription factor, Atf1-Pcr1, and other components of the transcriptional machinery.

Recruitment of P-TEFb (Cdk9-Pch1) to chromatin by the cap-methyl transferase Pcm1 in fission yeast.

Capping of nascent pre‐mRNAs is thought to be a prerequisite for productive elongation and associated serine 2 phosphorylation of the C‐terminal domain (CTD) of RNA polymerase II (PolII). The mechanism mediating this link is unknown, but is likely to include the capping machinery and P‐TEPb. We report that the fission yeast P‐TEFb (Cdk9‐Pch1) forms a complex with the cap‐methyltransferase Pcm1 and these proteins colocalise on chromatin. Ablation of Cdk9 function through chemical genetics causes growth arrest and abolishes serine 2 phosphorylation on the PolII CTD. Strikingly, depletion of Pcm1 also leads to a dramatic decrease of phospho‐serine 2. Chromatin immunoprecipitations show a severe decrease of chromatin‐bound Cdk9‐Pch1 when Pcm1 is depleted. On the contrary, Cdk9 is not required for association of Pcm1 with chromatin. Furthermore, compromising Cdk9 activity leads to a promoter‐proximal PolII stalling and sensitivity to 6‐azauracil, reflecting elongation defects. The in vivo data presented here strongly support the existence of a molecular mechanism where the cap‐methyltransferase recruits P‐TEFb to chromatin, thereby ensuring that only properly capped transcripts are elongated.

Fission yeast Csk1 is a CAK-activating kinase (CAKAK)

Cell cycle progression is dependent on the sequential activity of cyclin‐dependent kinases (CDKs). For full activity, CDKs require an activating phosphorylation of a conserved residue (corresponding to Thr160 in human CDK2) carried out by the CDK‐activating kinase (CAK). Two distinct CAK kinases have been described: in budding yeast Saccharomyces cerevisiae, the Cak1/Civ1 kinase is responsible for CAK activity. In several other species including human, Xenopus, Drosophila and fission yeast Schizosaccharomyces pombe, CAK has been identified as a complex homologous to CDK7–cyclin H (Mcs6–Mcs2 in fission yeast). Here we identify the fission yeast Csk1 kinase as an in vivo activating kinase of the Mcs6–Mcs2 CAK defining Csk1 as a CAK‐activating kinase (CAKAK).

F-box proteins: more than baits for the SCF?

Regulation of protein stability through the ubiquitin proteasome system is a key mechanism underlying numerous cellular processes. The ubiquitin protein ligases (or E3) are in charge of substrate specificity and therefore play a pivotal role in the pathway. Among the several different E3 enzyme families, the SCF (Skp1-Cullin-F box protein) is one of the largest and best characterized. F-box proteins, in addition to the loosely conserved F-box motif that binds Skp1, often carry typical protein interaction domains and are proposed to recruit the substrate to the SCF complex. Strikingly, genomes analysis revealed the presence of large numbers of F-box proteins topping to nearly 700 predicted in Arabidopsis thaliana.Recent evidences in various species suggest that some F-box proteins have functions not directly related to the SCF complex raising questions about the actual connection between the large F-box protein family and protein degradation, but also about their origins and evolution.

Mediator influences Schizosaccharomyces pombe RNA polymerase II-dependent transcription in vitro.

The fission yeast Schizosaccharomyces pombe has proved an important model system for cross-species comparative studies of many fundamental processes in the eukaryotic cell, such as cell cycle control and DNA replication. The RNA polymerase II transcription machinery is, however, still relatively poorly understood in S. pombe, partially due to the absence of a reconstituted in vitro transcription system. We have now purified S. pombe RNA polymerase II and its general initiation factors TFIIB, TFIIF, TFIIE, and TFIIH to near homogeneity. These factors enable RNA polymerase II to initiate transcription from the S. pombe alcohol dehydrogenase promoter (adh1p) when combined with Saccharomyces cerevisiae TATA-binding protein. We use our reconstituted system to examine effects of Mediator on basal transcription in vitro. S. pombe Mediator exists in two distinct forms, a free form, which contains the spSrb8, spTrap240, spSrb10, and spSrb11 subunits, and a smaller form, which lacks these four subunits and associates with RNA polymerase II to form a holoenzyme. We find that spSrb8/spTrap240/spSrb10/spSrb11 containing Mediator repress basal transcription, whereas Mediator lacking these subunits has a stimulatory effect on transcription. Our findings thus demonstrate that the spSrb8/spTrap240/spSrb10/spSrb11 subcomplex governs the ability of Mediator to stimulate or repress basal transcription in vitro.

Gene-specific requirement of RNA polymerase II CTD phosphorylation

The largest subunit of RNA polymerase II, Rpb1, contains an unusual C‐terminal domain (CTD) composed of numerous repeats of the YSPTSPS consensus sequence. This sequence is the target of post‐translational modifications such as phosphorylation, glycosylation, methylation and transitions between stereoisomeric states, resulting in a vast combinatorial potential referred to as the CTD code. In order to gain insight into the biological significance of this code, several studies recently reported the genome‐wide distribution of some of these modified polymerases and associated factors in either fission yeast (Schizosaccharomyces pombe) or budding yeast (Saccharomyces cerevisiae). The resulting occupancy maps reveal that a general RNA polymerase II transcription complex exists and undergoes uniform transitions from initiation to elongation to termination. Nevertheless, CTD phosphorylation dynamics result in a gene‐specific effect on mRNA expression. In this review, we focus on the gene‐specific requirement of CTD phosphorylation and discuss in more detail the case of serine 2 phosphorylation (S2P) within the CTD, a modification that is dispensable for general transcription in fission yeast but strongly affects transcription reprogramming and cell differentiation in response to environmental cues. The recent discovery of Cdk12 as a genuine CTD S2 kinase and its requirement for gene‐specific expression are discussed in the wider context of metazoa.

Cdk11-CyclinL Controls the Assembly of the RNA Polymerase II Mediator Complex

The large Mediator (L-Mediator) is a general coactivator of RNA polymerase II transcription and is formed by the reversible association of the small Mediator (S-Mediator) and the kinase-module-harboring Cdk8. It is not known how the kinase module association/dissociation is regulated. We describe the fission yeast Cdk11-L-type cyclin pombe (Lcp1) complex and show that its inactivation alters the global expression profile in a manner very similar to that of mutations of the kinase module. Cdk11 is broadly distributed onto chromatin and phosphorylates the Med27 and Med4 Mediator subunits on conserved residues. The association of the kinase module and the S-Mediator is strongly decreased by the inactivation of either Cdk11 or the mutation of its target residues on the Mediator. These results show that Cdk11-Lcp1 regulates the association of the kinase module and the S-Mediator to form the L-Mediator complex.

A coordinated codon-dependent regulation of translation by Elongator

More than a decade ago, the purification of the form of the RNA polymerase II (PolII) engaged in elongation led to the discovery of an associated, multi-subunit (Elp1-6) complex named “Elongator” by the Svejstrup lab. Although further evidence supported the original notion that Elongator is involved in transcription, Elongator lacked some of the expected features for a regulator of the elongating PolII. The discovery by the Byström lab, based on genetic dissection, that Elongator is pivotal for tRNA modifications, and that all the reported phenotypes of Elongator mutants are suppressed by the overexpression of two tRNAs added to the confusion. The increasing range of both potential substrates and biological processes regulated by Elongator in higher eukaryotes indicates that the major challenge of the field is to determine the biologically relevant function of Elongator. Our recent proteome-wide study in fission yeast supports a coordinated codon-dependent regulation of translation by Elongator. Here we provide additional analyses extending this hypothesis to budding yeast and worm.

Specificity of Cdk activation in vivo by the two Caks Mcs6 and Csk1 in fission yeast

Activating phosphorylation of cyclin‐dependent kinases (Cdks) is mediated by at least two structurally distinct types of Cdk‐activating kinases (Caks): the trimeric Cdk7–cyclin H–Mat1 complex in metazoans and the single‐subunit Cak1 in budding yeast. Fission yeast has both Cak types: Mcs6 is a Cdk7 ortholog and Csk1 a single‐subunit kinase. Both phosphorylate Cdks in vitro and rescue a thermosensitive budding yeast CAK1 strain. However, this apparent redundancy is not observed in fission yeast in vivo. We have identified mutants that exhibit phenotypes attributable to defects in either Mcs6‐activating phosphorylation or in Cdc2‐activating phosphorylation. Mcs6, human Cdk7 and budding yeast Cak1 were all active as Caks for Cdc2 when expressed in fission yeast. Although Csk1 could activate Mcs6, it was unable to activate Cdc2. Biochemical experiments supported these genetic results: budding yeast Cak1 could bind and phosphorylate Cdc2 from fission yeast lysates, whereas fission yeast Csk1 could not. These results indicate that Mcs6 is the direct activator of Cdc2, and Csk1 only activates Mcs6. This demonstrates in vivo specificity in Cdk activation by Caks.