Proteomic analysis of the U1 snRNP of Schizosaccharomyces pombe reveals three essential organism-specific proteins

Characterization of spliceosomal complexes in the fission yeast Schizosaccharomyces pombe revealed particles sedimenting in the range of 30–60S, exclusively containing U1 snRNA. Here, we report the tandem affinity purification (TAP) of U1-specific protein complexes. The components of the complexes were identified using (LC-MS/MS) mass spectrometry. The fission yeast U1 snRNP contains 16 proteins, including the 7 Sm snRNP core proteins. In both fission and budding yeast, the U1 snRNP contains 9 and 10 U1 specific proteins, respectively, whereas the U1 particle found in mammalian cells contains only 3. Among the U1-specific proteins in S. pombe, three are homolog to the mammalian and six to the budding yeast Saccharomyces cerevisiae U1-specific proteins, whereas three, called U1H, U1J and U1L, are proteins specific to S. pombe. Furthermore, we demonstrate that the homolog of U1-70K and the three proteins specific to S. pombe are essential for growth. We will discuss the differences between the U1 snRNPs with respect to the organism-specific proteins found in the two yeasts and the resulting effect it has on pre-mRNA splicing

Identification and transcriptional regulation of eukaryotic small nuclear RNA genes

L’attività di ricerca descritta in questo lavoro di tesi riguarda la regolazione trascrizionale di geni codificanti per small nuclear RNA (snRNAs), un gruppo di trascritti abbondanti, non codificanti proteine, che formano il cuore dello spliceosoma, deputato alla rimozione di introni dai pre-RNA messaggeri. Gli snRNA (U1-U6) sono sintetizzati dalla RNA Polimerasi (Pol) II, tranne lo snRNA U6, il cui gene è trascritto dalla RNA Polimerasi III. Rispetto agli snRNAs nei metazoi, si conosce poco della sintesi degli snRNA negli organismi unicellulari. Nel primo lavoro descritto in questa tesi abbiamo identificato putativi elementi regolatori nei promotori dei geni di lievito trascritti dalla Polimerasi II attraverso un “footprinting filogenetico”. L’analisi ha identificato alcuni blocchi conservati di sequenze corrispondenti ai consensi dei siti di legami per “general regulatory factors” (Rap1, Abf1, Reb1) ed alcuni elementi regolatori coinvolti in differenti pathways metabolici (RRPE - ribosomal RNA processing element; PACE - Proteasome Associated Control Element). Per capire più a fondo il coinvolgimento nella trascrizione dei putativi elementi regolatori così identificati, abbiamo preparato versioni marcate di due geni di S. cerevisiae codificanti per snRNA, LRS1 (codificante per U2 snRNA) e SNR7 (codificante per U5 snRNA), da utilizzare come geni reporter per analisi di espressione in vivo. L’analisi mutazionale degli elementi conservati nella regione a monte dell’inizio della trascrizione, ha rivelato la loro effettiva influenza nella trascrizione, probabilmente attraverso la creazione o il mantenimento di una regione libera da nucleosomi. La seconda parte di questa tesi si è focalizzata sulla caratterizzazione di nuove unità trascrizionali “tipo snRNA” nel genoma umano. Attraverso una ricerca computazionale per elementi tipici dei promotori dei geni per snRNAs (proximal sequence element and distal sequence element), abbiamo identificato alcune putative unità trascrizionali. Abbiamo analizzato le proprietà di trascrizione in vitro di alcune di queste unità in estratti nucleari da linee cellulari HeLa e SKNBE, mostrando come gli elementi promotori fossero effettivamente in grado di supportare la trascrizione da parte della Polimerasi III. In particolare abbiamo definito due nuovi RNA non codificanti, 17A e 51A, lunghi rispettivamente 171 e 423 nucleotidi, e la struttura dei promotori di altri due geni, 38A e 29A. Alcune delle unità trascrizionali caratterizzate (17A, 38A, 51A) sono interne a geni codificanti per proteine in zone dove avvengono fenomeni di splicing alternativo, mentre un’altra (29A) è stata identificata come una Alu. Ulteriori studi sono necessari per caratterizzare più in profondità tali unità trascrizionali in vivo e la funzione dei loro prodotti, in quanto la loro posizione suggerisce un possibile ruolo regolatorio ed una complessità crescente del trascrittoma della Polimerasi III.The research described in this thesis focused on transcriptional regulation of small nuclear RNA (snRNAs) genes, a group of highly abundant, non-protein-coding transcripts forming the core of the spliceosome, that catalyses the removal of introns from pre-mRNAs. SnRNAs (U1 to U6) are synthesized by RNA Polymerase (Pol) II, with the exception of the U6 snRNA, whose gene is transcribed by Pol III. As compared to the metazoan snRNAs, little is known about snRNA synthesis in unicellular organisms. In the first work described in this thesis we identified putative regulatory elements in yeast Pol II-transcribed snRNA gene promoters, starting from phylogenetic footprinting. The analysis revealed some evolutionarily conserved sequence blocks matching the consensus binding site of known general regulatory factors (Rap1, Abf1, Reb1) and a few regulatory elements known to be involved in different pathways (RRPE - ribosomal RNA processing element; PACE - Proteasome Associated Control Element). To better understand the involvement in transcription of the identified putative promoter elements we prepared sequence-tagged version of two S. cerevisiae snRNA genes, LRS1 (coding for U2 snRNA) and SNR7 (coding for U5 snRNA), to be used as reporter genes for in vivo expression analysis. Mutational analysis of the upstream conserved motifs revealed their influence on snRNA gene transcription, possibly involving the generation or maintenance of nucleosome-free promoter regions. The second part of this thesis focused on the characterization of novel snRNA gene-like transcriptional units in the human genome. By means of computer search for upstream promoter elements (proximal sequence element and distal sequence element) typical of small nuclear RNA genes, we have identified some putative transcription units. We analyzed the in vitro transcription properties in HeLa and SKNBE neuroblastoma cells nuclear extracts of some of these putative units, showing that their promoter elements were actually able to support Pol III-dependent transcription. In particular, we defined the boundary of two novel ncRNAs, 17A and 51A, 171 nt and 423 nt in length respectively and the promoter architectures of two other ncRNA genes, 38A and 29A. Some of the characterized transcriptional units (17A, 38A, 51A) are internal to known protein-coding genes where alternative splicing events takes place, while another (29A) was identified as an Alu. Further studies will be required to better characterize these transcription units in vivo and the function of their products, even though their location suggest a possible regulatory role and an increasing complexity of the pol III transcriptome

Processing of Non-Coding RNA by Mlp1 in Tetrahymena thermophila

RNA species are commonly transcribed as precursors and require post-transcriptional processing to become functional mature RNA transcripts. This includes the abundant cytoplasmic transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) that function in messenger RNA (mRNA) translation. Other non-coding RNAs also require extensive processing and assembly into ribonucleoproteins (RNPs), including small nuclear RNAs (snRNAs) that function in pre-mRNA splicing and small nucleolar RNAs (snoRNAs) involved in the processing and post-transcriptional modification of non-coding RNAs. The ciliate Tetrahymena thermophila is a highly studied eukaryotic model organism, however, many of its RNA processing pathways remain unexplored. In this work, we use molecular biology, biochemistry, cell biology and bioinformatic techniques to investigate the role of a novel La protein, Mlp1, in pre-tRNA, snRNP and snoRNP biogenesis. Unlike previously studied genuine La proteins, Mlp1 lacks an RNA-binding domain typically required for high-affinity binding of uridylate-tailed La target RNAs. We confirm that Mlp1 performs typical La protein functions, including uridylate-dependent preferential binding of pre-tRNAs and RNA chaperone activity to promote processing and maturation of misfolded nascent pre-tRNAs. However, in contrast to pre-tRNA processing in other eukaryotes, depletion of Mlp1 results in 3′-trailer stabilization instead of rapid trimming by 3′-exonucleases, indicating that Mlp1 is linked to a fundamentally different mechanism of tRNA processing in Tetrahymena thermophila. We also find that Mlp1 associates with mature snRNAs lacking the typical high affinity uridylate binding site and a core protein component of the snRNP. More specifically, Mlp1 interacts with the U4/U6 di-snRNP complex, which is formed during splicing, and Mlp1 depletion affects assembly of this complex. Mlp1 depletion also results in diminished splicing efficiency of pre-mRNAs, further supporting a functional role for Mlp1 in splicing in this system. Lastly, we show that Mlp1 associates with all previously annotated snoRNAs which function as guide RNAs in post-transcriptional modification of non-coding RNAs. We use this as a metric to predict novel snoRNAs in Tetrahymena thermophila and validate expression. Our work demonstrates new roles for the variant genuine La protein Mlp1 in the biogenesis of non-coding RNAs critical for the translation of proteins

Investigation of RNA-induced gene silencing in the fission yeast Schizosaccharomyces pombe

PhDRNA interference (RNAi) is a gene regulating system to which can silence genes at both transcriptional and post-transcriptional levels. In this thesis, a ura4-based RNAi selective assay was developed in the fission yeast Schizosaccharomyces pombe: antisense & sense constructs of mutually inverted ura4 DNA fragments were inserted under the regulation of a thiamine-repressible promoter; when induced by omission of thiamine, these transcripts triggered S. pombe ura4 gene silencing at the molecular level with the efficiency of -30%. Although dozens of proteins involved in RNAi pathways has been identified from different organisms, only a few are negative regulators of RNAi. The helF protein from the soil amoeba, Dictyostelium discoideum is one of the best characterized natural RNAi inhibitors. A homologue of Dictyostelium helF gene has also been identified in S. pombe. It is called. the mfhl gene. This thesis also addresses the question: is the mfhl gene a RNAi inhibitor in S. pombe. A haploid S. pombe mfhl deletion mutant was isolated, and the ura4-based RNAi selective assay was used to study RNAi in this mutant - there was no apparent effect on RNAi activity in this strain. Although no paralogue(s) of helF gene was found in Dictyostelium, a paralogue of S. pombe mfhl gene, mfh2 gene was identified -a mfhl & mf h2 double gene deletion mutant was also tested negative with respect to RNAi using the same assay. A phenotypic characterization of the S. pombe mfhl/mfh2 single/double gene deletion mutants compared to the wild type strain revealed that the mutants were more sensitive to ethanol, hydroxyurea and UV, which indicated that the mf hl and mf h2 genes appeared to protect S. pombe from chemical and UV induced DNA damage. The cellular localization of Dictyostelium helF and S. pombe mfhl genes was also attempted to be revealed by heterologous gene expressing studies using GFP-fusion expression plasmids in both Dictyostelium and S. pombe

The APL5 Subunit of the AP3 Adaptor Protein Complex is Required for Cytokinesis Checkpoint Function in Schizosaccharomyces Pombe

This study investigates the role of Apl5p in the complex regulatory network of Schizosaccharomyces pombe, which ensures the faithful and reliable completion of cytokinesis. This network, referred to as the cytokinesis checkpoint, ensures successful cell division upon perturbances to the cytokinetic machinery (e.g. disruption of the actin cytoskeleton). Apl5p has been identified as a putative regulator of the cytokinesis checkpoint based on the hyper-sensitivity of apl5D mutants to the actin depolymerizing drug, Latrunculin A. Apl5p is an essential subunit of the conserved AP3 adaptor complex, which is suspected to be involved in vesicular trafficking. Thus, I hypothesized that Apl5p mediates the transport of materials which are necessary for cytokinetic regulation during stress. In this report, I show that apl5D mutants are inviable in the presence of LatA due to their inability to complete cytokinesis. Using live-cell imaging, I show that the mutant’s failure to complete cytokinesis results from an underlying inability to maintain the physical integrity of the actomyosin ring upon the initiation of constriction. Over-expression of Apl5p resulted in a dominant negative effect and impeded cell viability upon treatment of LatA (rather than conferring LatA resistance). Lastly, I determined the intracellular localization of Apl5p by monitoring Apl5-YFP fusion proteins. Given its role in vesicular trafficking, Apl5p was expected to localize to cytoplasmic vesicles at, or near, the site of cell division. Surprisingly, Apl5p-YFP fusion proteins were instead found to localize to small punctate structures within, or on, the cell nucleus. This contradicts both my hypothesis and our current understanding of the functions of the AP3 complex. Therefore, further research is necessary to determine the role of Apl5p in cytokinetic regulation, especially concerning its localization