SUMMARY Messenger RNA decay measurements are typically performed on a population of cells. However, this approach cannot reveal sufficient complexity to provide information on mechanisms that may regulate mRNA degradation, possibly on short time scales. To address this deficiency, we measured cell cycle regulated decay in single yeast cells using single-molecule FISH. We found that two genes responsible for mitotic progression, SWI5 and CLB2 exhibit a mitosis-dependent mRNA stability switch. Their transcripts are stable until mitosis when a precipitous decay eliminates the mRNA complement, preventing carry-over into the next cycle. Remarkably, the specificity and timing of decay is entirely regulated by their promoter, independent of specific cis mRNA sequences. The mitotic exit network protein, Dbf2p binds to SWI5 and CLB2 mRNAs co-transcriptionally and regulates their decay. This work reveals the promoter-dependent control of mRNA stability, a novel regulatory mechanism that could be employed by a variety of mRNAs and organisms.

AGRADECIMIENTOS vi viiDurante toda esta tesis veía clarísimo todo lo que escribir en este apartado y ahora que llega el momento me quedo en blanco... Debe ser la presión de saber que va a ser la sección más leída… Primero agradecer a José y a Elena el poder haber trabajado en este laboratorio durante este tiempo. Han sido casi dos tesis, sí, sí, mi primera página del cuaderno de laboratorio data del 19 de Abril del 2000, todavía recuerdo el primer día, lo primero que hice fue rotular eppendorfs con una temblorosa mano, cuántos nervios. Después de trabajar en muchos temas, momentos difíciles y agradables, odiosos y entrañables, mudanzas, meetings, reuniones, cenas y mil otros recuerdos que me llevo puedo decir que me va a costar estar lejos de este lugar. Un abrazo muy fuerte a los dos!! Mis compañeros de laboratorio, pasados, presentes y futuros, desde aquel chico de la UAB que me enseñó como utilizar una enzima de restricción hasta el chico nuevo que todavía no conozco... No tengo palabras. Habéis sido mi familia todo este tiempo:A la pequeña Ester que me ha cuidado desde el principio como una hermana mayor. A Ana con la que el odio y el cariño se entrelazaban continuamente, muy buena conversadora y con gran ojo crítico. A Carine cuya breve estancia me dejó entrever que hay más de una manera de hacer ciencia. A Jordi (MalaP), compañero de adversidades y aventuras. A Mercè, cuyo apoyo logístico y moral ha sido imprescindible. A Alice, símbolo de la ternura y de la filología italiana, ya se sabe, a caballo donato... A Miriam, una de las mejores personas que conozco, sensata, seria y muy inteligente, una gran científica. A Mónica, maestra de artes marciales y colis. A Blanca, la dulzura personificada y buen público para los chistes. A Nati, también compañera de poyata, penas y alegrías, una post-doc todavía sin título. A Isabel, la catol... una gran amiga y compañera de ChIPs, tranquila que las cosas se van arreglando solas! A Sarela, un meiga pero de las buenas y a Iván (el granaíno), que con su corta pero intensa estancia nos alegró las largas horas de laboratorio. A todos gracias por apoyarme, aguantarme y porque nadie mejor que vosotros sabe lo que supone esta tesis.Al resto de gente de la UPF, los vecinos de laboratorio, especialmente el laboratorio de Señalización Celular (o cariñosamente llamados "los posas") con los que hemos compartido más que un laboratorio, Inmunología (Mari, Bea, Mingui, Julia, Rosa, etc.), grandes amigos dentro y fuera de la UPF, y todos los demás grupos del departamento.Siguiendo la ampliación de espacio, gracias también a toda la gente del PRBB que han ayudado a que estos años se hicieran más amenos. Gracias a ellos se materializó viii la mejor terapia antiestrés, el voley, gracias a los Rabenessen de Caramel (incluido Iván, sí, podrás venir al pica pica de mi tesis, jeje).Gracias a mis compañeros de licenciatura, con ellos inicié este camino y espero seguir sendas cercanas a las suyas.A la gente del laboratorio de Paul Nurse en New York, gracias por haberme hecho pasar una de las m...

Summary During the last decade, much has been learnt about the mechanisms by which oxidative stress is perceived by aerobic organisms. The Schizosaccharomyces pombe Pap1 protein is a transcription factor localized at the cytoplasm, which accumulates in the nucleus in response to different inducers, such as the pro‐oxidant hydrogen peroxide (H2O2) or the glutathione‐depleting agent diethylmaleate (DEM). As described for other H2O2 sensors, our genetic data indicates that H2O2 reversibly oxidizes two cysteine residues in Pap1 (Cys278 and Cys501). Surprisingly, our studies demonstrate that DEM generates a non‐reversible modification of at least two cysteine residues located in or close to the nuclear export signal of Pap1 (Cys523 and Cys532). This modification impedes the interaction of the nuclear exporter Crm1 with the nuclear export signal located at the carboxy‐terminal domain of Pap1. Mass spectrometry data suggest that DEM binds to the thiol groups of the target cysteine residues through the formation of a thioether. Here we show that DEM triggers Pap1 nuclear accumulation by a novel molecular mechanism.

Mutations in the U2 snRNP component SF3B1 are prominent in myelodysplastic syndromes (MDSs) and other cancers and have been shown recently to alter branch site (BS) or 3 ′ splice site selection in splicing. However, the molecular mechanism of altered splicing is not known. We show here that hsh155 mutant alleles in Saccharomyces cerevisiae, counterparts of SF3B1 mutations frequently found in cancers, specifically change splicing of suboptimal BS pre-mRNA substrates. We found that Hsh155p interacts directly with Prp5p, the first ATPase that acts during spliceosome assembly, and localized the interacting regions to HEAT (Huntingtin, EF3, PP2A, and TOR1) motifs in SF3B1 associated with disease mutations. Furthermore, we show that mutations in these motifs from both human disease and yeast genetic screens alter the physical interaction with Prp5p, alter branch region specification, and phenocopy mutations in Prp5p. These and other data demonstrate that mutations in Hsh155p and Prp5p alter splicing because they change the direct physical interaction between Hsh155p and Prp5p. This altered physical interaction results in altered loading (i.e., "fidelity") of the BS-U2 duplex into the SF3B complex during prespliceosome formation. These results provide a mechanistic framework to explain the consequences of intron recognition and splicing of SF3B1 mutations found in disease.

The meiotic cell cycle is modified from the mitotic cell cycle by having a premeiotic S phase which leads to high levels of recombination, a reductional pattern of chromosome segregation at the first division, and a second division with no intervening DNA synthesis. Cyclin-dependent kinases are essential for progression through the meiotic cell cycle, as for the mitotic cycle. Here we show that a fission yeast cyclin, Rem1, is present only during meiosis. Cells lacking Rem1 have impaired meiotic recombination, and Rem1 is required for premeiotic DNA synthesis when Cig2 is not present. rem1 expression is regulated at the level of both transcription and splicing, with Mei4 as a positive and Cig2 a negative factor of rem1 splicing. This regulation ensures the timely appearance of the different cyclins during meiosis, which is required for the proper progression through the meiotic cell cycle. We propose that the meiosis-specific B-type cyclin Rem1 has a central role in bringing about progression through meiosis.During its life cycle, the fission yeast Schizosaccharomyces pombe can undergo either mitotic proliferation or sexual conjugation followed by meiosis. The decision between these two developmental fates occurs in the G 1 phase of the cell cycle. Fission yeast cells proliferate in a haploid state, and when the nitrogen source becomes limiting they arrest in G 1 and conjugate with cells of the opposite mating type (11, 37). The pathway controlling entry into meiosis is quite well understood in S. pombe. Nitrogen starvation induces the expression of several genes, including mei2, which encodes an RNA-binding protein that is inactivated during mitotic growth by direct phosphorylation by the protein kinase Pat1 (25,35,36), and mei3, which encodes an inhibitor of Pat1 protein kinase (26). The temperature-sensitive pat1-114 allele initiates meiosis at the restrictive temperature (17,25,26,29) and can be used to synchronously induce meiosis, even in haploid cells.When diploid zygotes proceed into meiosis, they transiently arrest in G 1 and then initiate one round of DNA replication (premeiotic S phase), leading to cells with a 4C DNA content. Replication is followed by high levels of recombination, chromosome pairing, and two consecutive nuclear divisions, generating four nuclei with a 1C DNA content (for a review, see reference 38). Premeiotic S phase takes longer than mitotic S phase, although, at least in Saccharomyces cerevisiae, the same replication origins are used and the replication forks move at the same rate (8). Although many gene products essential for mitotic DNA synthesis are also required for premeiotic S phase (28), there are some exceptions. For example, in S. cerevisiae, two S-phase cyclins, CLB5 and CLB6, are not required for completion of mitotic DNA synthesis but are essential for premeiotic S phase (34). These differences between mitotic and meiotic DNA synthesis might be related to the period of high recombination that follows premeiotic S phase. In fact, DSBs (double-strand breaks) and thus me...