33 research outputs found

    Insights into RNA Splicing and the Regulation of Gene Expression in Saccharomyces cerevisiae

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    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

    Strategies for developing a benchtop biosensor for the rapid detection of L-Alanine

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    As the subtle intricacies of mammalian metabolism have become further elucidated over the past several decades, substantial progress has been made in the medical and scientific communities to address metabolic disorders and the relationships between abnormalities in metabolism at the molecular level and their observable physiological ramifications. The amino acid alanine is known as a key molecule in protein and carbohydrate metabolism and nitrogen regulation, and has further been associated with a number of diseases and pathological conditions. It is also of interest to the commercial and biotechnological industries. Therefore, a rapid and inexpensive procedure for the measurement of alanine in biological or commercial samples would be of great use. Two amperometric methods for the measurement of L-alanine involving enzymatic biosensors are explored. The electrochemical foundations for the operation of the sensors are described. The unique reaction mechanism for the conversion of alanine to measurable products is discussed. Sensors are tested for alanine measurement via the oxidation of hydrogen peroxide and the reduction of oxygen. The response of the sensors to reacted alanine samples as well as standard solutions are evaluated with regards to sensitivity and rapidity. These studies show that the proposed reaction mechanism for alanine conversion and detection is feasible and that the development of an amperometric biosensor for this purpose is an attainable goal, while also suggesting that optimizing the sensitivity of the sensors is paramount to its utilit

    Splicing-Mediated Autoregulation Modulates Rpl22p Expression in <i>Saccharomyces cerevisiae</i>

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    <div><p>In <i>Saccharomyces cerevisiae</i>, splicing is critical for expression of ribosomal protein genes (RPGs), which are among the most highly expressed genes and are tightly regulated according to growth and environmental conditions. However, knowledge of the precise mechanisms by which RPG pre-mRNA splicing is regulated on a gene-by-gene basis is lacking. Here we show that Rpl22p has an extraribosomal role in the inhibition of splicing of the <i>RPL22B</i> pre-mRNA transcript. A stem loop secondary structure within the intron is necessary for pre-mRNA binding by Rpl22p <i>in vivo</i> and splicing inhibition <i>in vivo</i> and <i>in vitro</i> and can rescue splicing inhibition <i>in vitro</i> when added in <i>trans</i> to splicing reactions. Splicing inhibition by Rpl22p may be partly attributed to the reduction of co-transcriptional U1 snRNP recruitment to the pre-mRNA at the <i>RPL22B</i> locus. We further demonstrate that the inhibition of <i>RPL22B</i> pre-mRNA splicing contributes to the down-regulation of mature transcript during specific stress conditions, and provide evidence hinting at a regulatory role for this mechanism in conditions of suppressed ribosome biogenesis. These results demonstrate an autoregulatory mechanism that fine-tunes the expression of the Rpl22 protein and by extension Rpl22p paralog composition according to the cellular demands for ribosome biogenesis.</p></div

    Model for the autoregulation of <i>RPL22B</i>.

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    <p><i>RPL22B</i> pre-mRNA contains an intronic regulatory element, depicted here by a simplified stem loop, to which the Rpl22 protein is able to directly or indirectly bind to associate with the unspliced mRNP. This association inhibits splicing of the pre-mRNA, potentially by prohibiting binding of one or more spliceosomal snRNPs, which is then either degraded by nuclear exonucleases or exported to the cytoplasm and degraded by NMD. Splicing inhibition is exacerbated during DNA damage stress. The green protein labeled with the black question mark represents potential binding factors that may facilitate an indirect interaction between Rpl22p and the <i>RPL22B</i> unspliced mRNP.</p

    Rpl22Ap modulates the splicing of <i>RPL22B</i> pre-mRNA, which is targeted for degradation by cytoplasmic and nuclear decay factors.

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    <p><b>A.</b> RT-PCR analysis of splicing products of the <i>RPL22B</i> pre-mRNA in wild-type and ribosomal protein deletion mutants. Bands indicate the unspliced (US), alternatively-spliced (*AS 5’), and spliced (S) species. <b>B.</b> RT-PCR analysis of splicing products of the <i>TFC3</i> pre-mRNA in wild-type and ribosomal protein deletion mutants. Bands indicate the unspliced (US), spliced (S), and alternatively spliced (*AS 3’) species. <b>C.</b> Northern blot analysis of <i>RPL22B</i> splicing products in wild-type, <i>upf1</i>Δ, <i>rpl22a</i>Δ, and <i>upf1</i>Δ/<i>rpl22a</i>Δ cells detected using an <i>RPL22B</i> 5’UTR riboprobe. Shown are the unspliced (US) and spliced (S) species. <i>SCR1</i> was used as a loading control. <b>D.</b> Northern blot showing <i>RPL22B</i> splicing products in wild-type or <i>upf1</i>Δ cells with an empty pUG23 vector or intronless <i>RPL22A</i> cDNA overexpression plasmid. Splicing products were detected using an <i>RPL22B</i> 5’UTR riboprobe. Labeling of the transcripts is similar to panel C. <i>SCR1</i> was used as a loading control. <b>E.</b> Northern blot analysis of <i>RPL22B</i> in cytoplasmic RNA decay mutants carrying the multi-copy YEp24 vector with either no insert or expressing the intronless <i>RPL22A</i> gene. Labeling of the transcripts is similar to panels C-D. <i>GAPDH</i> was used as a loading control. <b>F.</b> Northern blot analysis of <i>RPL22B</i> in nuclear RNA decay mutants carrying an empty YEp24 vector or an intronless <i>RPL22A</i> overexpression plasmid. Strains were grown to exponential phase in–URA media at 25°C and then shifted to 37°C for three hours. Labeling of the transcripts is similar to panels C-E. <i>SCR1</i> was used as a loading control. <b>G.</b> Northern blot analysis of <i>RPL22B</i> in <i>xrn1</i>Δ and NMD mutant strains carrying an empty YEp24 vector or an <i>RPL22A</i> overexpression plasmid. Detection methods and labeling are similar to panels C-F. <i>GAPDH</i> was used as a loading control.</p
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