Author: Katherine Anne Senn
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
Get Book Here
Book Description
Pre-mRNA splicing, the removal of noncoding introns to form a protein coding mRNA, is a crucial step in eukaryotic gene expression. The spliceosome, a macromolecular complex made up of five small nuclear RNAs (snRNAs) and dozens of proteins organized into small nuclear ribonucleoproteins (snRNPs), catalyzes splicing in two steps. These steps are highly regulated to ensure correct mRNA isoform production and protein expression. Core components of the spliceosome as well as additional auxiliary factors tune splicing for accuracy and regulation of alternative splicing outcomes. Because of the complexity of splicing regulation, there are many open questions about the factors involved and the regulation of alternative splicing. This thesis addresses the functions of two yeast proteins involved in splicing regulation and an alternatively spliced gene in humans.In chapters 2 and 3, I discuss the novel yeast splicing factor Fyv6. Genetic and biochemical evidence supports a role for Fyv6 in the second step of splicing, exon ligation. A collaborator has determined the structure of a Fyv6-containing spliceosome by cryo-electron microscopy. Using this structure, we have used biochemical and transcriptomics experiments to elucidate how Fyv6 affects 3' splice site choice. Yeast splicing regulatory factor Mer1 interacts with introns and the U1 snRNP to enhance splicing of a subset of meiotically-regulated pre-mRNAs by an as-yet undetermined mechanism. With the goal of elucidating the mechanism, I recapitulated Mer1-dependent splicing in vitro and used colocalization single molecule microscopy to examine how Mer1 affects the dynamics of U1 binding to pre-mRNAs (Chapter 4). Our results indicate Mer1 promotes splicing without significantly altering the observed distributions of U1 binding events. This study lays the groundwork for future studies of splicing regulation by Mer1 and other factors. Finally, I studied the expression and alternative splicing of human glucose transporter family member GLUT8 (Chapter 5). Multiple alternatively spliced GLUT8 mRNA isoforms exist, but only some encode viable proteins. One that does not is upregulated in cancers, which reduces GLUT8 protein expression. Collaborators showed that based on its localization, inability to transport glucose, and release of a cleaved peptide, the main functional isoform of GLUT8 may be a metabolic sensor.
Author: Jason Gabunilas
Publisher:
ISBN:
Category :
Languages : en
Pages : 175
Get Book Here
Book Description
RNA splicing is a critical component in the regulation of gene expression in all eukaryotes. The work described herein chronicles our investigative efforts into three facets of RNA splicing and their associated mechanisms in the model organism Saccharomyces cerevisiae. Previous work from our group highlighted the ability for nonsense-mediated mRNA decay (NMD) to mask the splicing defects of splicing factor mutants, suggesting that the full repertoire of splicing substrates and products is at least partly occluded by RNA surveillance mechanisms. Continuing this work, we sought to uncover previously unidentified splicing events by performing RNA-sequencing in wild-type yeast as well as strains deficient in NMD. This analysis revealed that alternative splicing at unannotated non-canonical 5'- and 3'-splice sites occurs within a large number pre-mRNAs in yeast, but that these events are not usually observed because they introduce premature termination codons (PTCs) into the translational reading frames of the spliced transcripts, thereby rendering them targets for degradation by NMD. This work demonstrated that the degree of alternative splicing in yeast RNA transcripts is greater than previously appreciated, and that alternative splicing linked to NMD (AS-NMD) serves to regulate overall transcript levels. Notably, this study uncovered a non-productive alternative 5'-splice site for the ribosomal protein gene RPL22B that is activated in a stress-dependent manner. We further investigated the splicing of RPL22B and found that its protein product Rpl22p functions in an extra-ribosomal capacity by inhibiting the splicing of its own pre-mRNA, defining an autoregulatory splicing-mediated negative feedback mechanism that fine-tunes the expression of Rpl22p with potential implications in stress response. Finally, we identified a global role for the second-step splicing factor Prp18p in the suppression of non-canonical alternative 3'-splice sites throughout the yeast transcriptome. Specifically, we found that branchpoint-proximal alternative 3'-splice sites are activated in the absence of Prp18p in a substantial fraction of intron-containing genes. These results suggest that Prp18p is responsible for maintaining the fidelity of RNA splicing. Together, these studies reveal new insights into gene regulation by highlighting the interplay between RNA splicing and quality control.
Author: Keiko Sakai
Publisher:
ISBN:
Category : RNA splicing
Languages : en
Pages : 438
Get Book Here
Book Description
Author: Laura-Oana Albulescu
Publisher:
ISBN:
Category :
Languages : en
Pages : 320
Get Book Here
Book Description
Pre-mRNA splicing is an essential eukaryotic pathway which controls gene expression. Increasing lines of evidence indicate links between splicing and other RNA processing pathways such as chromatin remodeling, transcription and 3'end processing, yet in many cases the specific proteins responsible for functionally connecting these pathways remain unclear. To determine the full complement of factors which impact pre-mRNA splicing, I developed a genome-wide screen in Saccharomyces cerevisiae which allowed me to evaluate differences in splicing efficiency in the background of ~5500 unique gene mutations. By measuring expression changes in precursor levels by high-throughput quantitative PCR, I detected enrichment in several classes of genes, with very strong candidates mapping to the chromatin remodeling, transcription and 3'end processing classes. One of these candidates is the bromodomain protein Bdf1, a component of the transcription factor TFIID and also a member of the SWR-C chromatin remodeling complex. Splicing sensitive microarrays confirm that deletion of Bdf1 leads to a global splicing defect, while ChIP-qPCR data reveal a decrease in U1 snRNP recruitment at intron containing genes, suggesting an inhibitory effect on spliceosome assembly. Conversely, Bdf1's homologue Bdf2 with which it is 46% identical, does not impact pre-mRNA splicing or spliceosome recruitment, consistent with my hypothesis that Bdf2 functions mainly in transcription. To further characterize Bdf1 function, I modified the high-throughput screening approach described above and employed it in a forward genetic manner to enable a mutagenic analysis of the Bdf1 protein. This analysis revealed that the C-terminal tail which overlaps with the Taf7 interaction domain, and contains a conserved SEED region and one of the known phosphorylation sites in Bdf1, may be responsible for the splicing defect. In opposition to the global splicing defect exhibited by Bdf1, mutations in 3'end processing factors such as Cft2 and Yth1 result in transcript-specific defects. My results highlight the cross-talk between 5' and 3'end processing factors and the spliceosome, and support a model in which the definition of terminal exons in the budding yeast is identical with the mechanism described in higher systems. Furthermore, the novel role of Bdf1 at the interface of transcription and pre-mRNA splicing suggests a new mechanism that underlies the coupling between these two RNA pathways.
Author: Klemens J. Hertel
Publisher: Humana
ISBN: 9781627039796
Category : Medical
Languages : en
Pages : 0
Get Book Here
Book Description
Providing a guide to classical experimental approaches to decipher splicing mechanisms and experimental strategies that rely on novel multi-disciplinary approaches, Spliceosomal Pre-mRNA Splicing: Methods and Protocols describes the theory of alternative pre-mRNA splicing in seven introductory chapters and then introduces protocols and their theoretical background relevant for a variety of experimental research. These protocol chapters cover basic methods to detect splicing events, analyses of alternative pre-mRNA splicing in vitro and in vivo manipulation of splicing events and high-throughput and bioinformatic analyses of alternative splicing. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols and tips on troubleshooting and avoiding known pitfalls. Comprehensive and practical, Spliceosomal Pre-mRNA Splicing: Methods and Protocols will aid newcomers and seasoned molecular biologists in understanding the fascinating world of alternative splicing with the ultimate goal of paving the way for many new discoveries to come.
Author: Usha Vijayraghavan
Publisher:
ISBN:
Category : Electronic dissertations
Languages : en
Pages : 370
Get Book Here
Book Description
Author: Javier Armisen Garrido
Publisher:
ISBN:
Category : Biochemistry
Languages : en
Pages : 155
Get Book Here
Book Description
In eukaryotes, genes are presented in a series of coding and non-coding DNA regions (exons/introns) that are transcribed into a premature RNA (pre-mRNA). Introns can be removed from the mature premRNA, before its translation into proteins, in a process called splicing. The splicing reaction occurs in two highly regulated transesterification reactions inside of the cell nucleus, and it is catalyzed by the Spliceosome, involving the binding and release of five small nuclear ribonucleoprotein particles (snRNPs). While some introns are constitutively spliced, others can be alternatively spliced, giving different exon combinations and therefore different proteins, increasing the protein diversity of the species. In humans, misregulation of alternative splicing can result in the production of aberrant proteins, some of which may produce cancer or other severe diseases. In yeast, alternative splicing is regulated by different splicing factors, such as Mer1p. Mer1p is expressed during meiosis in the yeast Saccharomyces cerevisiae and activates the splicing in at least three different genes (AMA1, MER2, and MER3), which contain a conserved intronic splicing enhancer sequence. Previous results have shown that Mer1p is able to interact with the pre-mRNA and with specific proteins of the U1 and U2 snRNPs. However, the specific molecular mechanisms by which Mer1p activates splicing remained unknown. The objective of this work is to determine how Mer1p regulates the splicing of its targets, and how different splicing factors modulate Mer1p activity. Using biochemistry and genetics, the data presented in this work indicate that Mer1p recruits the snRNPs U1, U2 and U6, to pre-mRNA. This recruitment of the snRNPs is dependent of the U1 snRNP protein Nam8p and the U2 snRNP protein Snu17p, but independent on the branchpoint region or ATP. Furthermore, Mer1p accelerates and stabilizes the formation of the early complexes of the spliceosome. Finally, U1 and U2 are recruited to the pre-mRNA at the same time, emerging a new alternative hypothesis of splicing regulation that can be applied to other enhancer regulators and that differs from the classical model of stepwise assembly of the snRNP.
Author: Frederick W. Scherrer (Jr.)
Publisher:
ISBN:
Category : Cell division
Languages : en
Pages : 107
Get Book Here
Book Description
The transition from mitosis to meiosis in the yeast Saccharomyces cerevisiae requires a significant change to gene expression profiles. Regulation of pre-messenger RNA splicing patterns during meiosis assists in this transition by fine tuning expression of essential meiotic genes. Produced only during meiosis, Mer1p is linked to the splicing of at least three mRNAs: MER2, MER3, and AMA1. Previous evidence suggests that Mer1p activates splicing by directly recruiting snRNPs or stabilizing intermediate splicing complexes formed on pre-mRNA that contains an intronic Mer1p enhancer element. However, some splicing factors, especially accessory/non-snRNP factors, have critical roles in retaining unspliced pre-mRNAs in the nucleus. I tested if Mer1p may indirectly regulate splicing by preventing the export of pre-mRNAs to the cytoplasm and also demonstrated that a second subunit of the Retention and Splicing (RES) complex, Bud13p, has transcript-specific effects on Mer1p-activated splicing. The results indicated that Mer1p can retain unspliced pre-mRNA in the nucleus; however, nuclear retention could not be uncoupled from splicing activation. In the absence of Mer1p, the AMA1 pre-mRNA is exported to the cytoplasm, translated, but not subjected to nonsense-mediated decay (NMD) despite a premature stop codon in the intron. A novel role for the Mer1p activation domain was revealed by a two-hybrid interaction with Prp39p, an essential U1 snRNP protein. This suggests the initial contact between Mer1p and the spliceosome occurs during commitment complex assembly. Collectively, these data imply that Mer1p can retain pre-mRNAs in the nucleus only by facilitating their interaction with the spliceosome and support models for cytoplasmic degradation of unspliced pre-mRNAs that fail to assemble into spliceosomes in yeast. A two-hybrid analysis of U1 snRNP proteins and other early splicing factors tested 460 possible interactions and the several novel interactions reported here indicate a revised model for U1snRNP structure.
Author: Witold Filipowicz
Publisher: Springer Science & Business Media
ISBN: 9400903537
Category : Science
Languages : en
Pages : 419
Get Book Here
Book Description
A recent volume of this series (Signals and Signal Transduction Pathways in Plants (K. Palme, ed.) Plant Molecular Biology 26, 1237-1679) described the relay races by which signals are transported in plants from the sites of stimuli to the gene expression machinery of the cell. Part of this machinery, the transcription apparatus, has been well studied in the last two decades, and many important mechanisms controlling gene expression at the transcriptional level have been elucidated. However, control of gene expression is by no means complete once the RNA has been produced. Important regulatory devices determine the maturation and usage of mRNA and the fate of its translation product. Post-transcriptional regulation is especially important for generating a fast response to environmental and intracellular signals. This book summarizes recent progress in the area of post-transcriptional regulation of gene expression in plants. 18 chapters of the book address problems of RNA processing and stability, regulation of translation, protein folding and degradation, as well as intracellular and cell-to-cell transport of proteins and nucleic acids. Several chapters are devoted to the processes taking place in plant organelles.
Author: Baldev K. Vig
Publisher: Springer Science & Business Media
ISBN: 3642849385
Category : Science
Languages : en
Pages : 432
Get Book Here
Book Description
Proceedings of the NATO Advanced Research Workshop on Chromosome Segregation and Aneuploidy, held at Aghia Pelagia, Greece, October 10-15, 1992
