Biology subjects

Fenton, D. A.

Publications and source records attributed to Fenton, D. A..

4 recordsLinked to original sources

bioRxiv2025

Comprehensive analysis of yeast +1 ribosomal frameshifting unveils a novel stimulator affirming two distinct frameshifting mechanisms

Ribosomal frameshifting is an important, albeit rare, mRNA decoding mechanism that generally allows the synthesis of a single protein from two different reading frames. For most +1 frameshifting cases, the mechanism is commonly presumed to involve dissociation of the P-site tRNA from its cognate codon followed by its movement to the +1 codon, setting the new +1 frame for incoming tRNAs. This movement is stabilized by P-site tRNA pairing with the +1 codon. However, in several occurrences in the yeast Saccharomyces cerevisiae, P-site tRNA re-pairing with the +1 codon is impossible. Two alternative hypotheses exist explaining this observation. One model suggests that +1 frameshifting occurs according to a common mechanism involving P-site movement, while its re-pairing with +1 codon is not essential. The alternative model suggests a distinct mechanism in which the A-site tRNA acceptance at the +1 codon occurs in the absence of P-site tRNA movement relative to mRNA. Here we set out to perform a comprehensive comparative analysis of all known +1 ribosomal frameshifting sites in S. cerevisiae. This included a novel case of +1 ribosomal frameshifting that we discovered during this study. It is required for the expression of LLP1 gene encoding dolichol-linked oligosaccharide pyrophosphatase. During the analysis of all frameshifting contexts, we identified a conserved RNA secondary structure located almost immediately upstream of the ABP140 frameshifting site. This structure substantially increases +1 frameshifting efficiency. The RNA stimulators location suggests that mRNA exiting the ribosome forms this structure, creating an mRNA pulling effect, thus favouring positioning of the +1 codon in the P-site. Placing the stimulator upstream of various known frameshifting sites, revealed that its stimulatory action is selective to those frameshifting sites where P-site tRNA re-pairing is possible, reinforcing the idea of two distinct mechanisms of ribosomal frameshifting.

molecular biology↗

bioRxiv2022

Regulated use of alternative Transcription Start Sites controls the production of cytosolic or mitochondrial forms of branched-chain aminotransferase in Kluyveromyces marxianus

Following a whole genome duplication (WGD) event approximately 100 million years ago, the yeast lineage from which the model Saccharomyces cerevisiae derives maintained two copies of genes where it was necessary to synthesise proteoforms with different sub-cellular localisation. In contrast, yeasts that did not undergo the WGD event have a single gene that must encode both proteoforms. We adopted an integrated in silico and experimental approach to study how this is achieved with BAT1, a gene that encodes mitochondrial and cytosolic forms of a branched chain aminotransferase (BCAT) in pre-WGD yeast such as Kluyveromyces marxianus. We determined that condition-specific regulation of alternative transcription sites gives rise to mRNA isoforms that differ at the 5end and that, when decoded, generate a mitochondrial or cytosolic proteoform. Furthermore, targeted mutants lacking specific transcription factors were generated to establish how this differentiation was regulated. As in S. cerevisiae, Gcn4 and Leu3 activated expression of the mRNA encoding the mitochondrial proteoform under conditions when branched chain amino acid synthesis was required. Unlike S. cerevisiae, however, K. marxianus lacked tight regulation of the mRNA encoding the cytosolic proteoform supporting the hypothesis that maintaining paralogous genes in post-WGD yeasts facilitated development of more sophisticated expression control mechanisms.

genetics↗

bioRxiv2022

Integrated data-driven reannotation of the Kluyveromyces marxianus genome reveals an expanded protein coding repertoire

The coding potential of the eukaryotic genome can be greatly expanded by the regulated use of mechanisms that generate more than one protein product from a gene. We combined techniques for mapping 5 and 3 ends of RNA transcripts with ribosome profiling to study the organisation of protein coding gene expression in the yeast Kluyveromyces marxianus. We uncovered over 1000 cases of novel proteoforms due to use of alternative transcription or translation start sites, identified 800 translated upstream open reading frames, observed surprising translation of antisense RNAs, and discovered a novel case of programmed ribosomal frameshifting. In some cases, features are conserved across yeast species, whereas others are species-specific. This offers new possibilities to explore the evolution of genomes and gene regulation in budding yeasts. Our analysis also enabled us to improve the genome annotation of K. marxianus by adding or correcting annotations of over 300 protein coding genes. The processed data has been made available on the GWIPS-viz and Trips-Viz browsers, thus providing an accurate data-driven annotation of transcripts and their protein coding regions along with quantitative information on their transcription and translation.

genomics↗

bioRxiv2022

Development of a Ribosome Profiling Protocol to Study Translation in the yeast Kluyveromyces marxianus

Kluyveromyces marxianus is an interesting and important yeast because of particular traits like thermotolerance and rapid growth, and applications in food and industrial biotechnology. Knowing how K. marxianus responds and adapts to changing environments is important to achieve a full understanding of the its biology and to develop bioprocesses. For this, a full suite of omics tools to measure and compare global patterns of gene expression and protein synthesis is needed. Whereas transcriptome analysis by RNA-Seq quantifies mRNA abundance, ribosome profiling allows codon-resolution of translation on a genome-wide scale by deep sequencing of ribosome locations on mRNAs and is emerging as a valuable tool to study translation control of gene expression. We report here the development of a ribosome profiling method for K. marxianus and we make the procedure available as a step by step protocol. To aid in the analysis and sharing of ribosome profiling data, we also added the K. marxianus genome as well as transcriptome and ribosome profiling data to the publicly accessible GWIPS-viz and Trips-Viz browsers. Users are able to upload custom ribosome profiling and RNA-Seq data to both browsers, therefore allowing easy analysis and sharing of data. As many studies only focus on the use of RNA-Seq to study K. marxianus in different environments, the availability of ribosome profiling is a powerful addition to the K. marxianus toolbox. Graphical abstractDevelopment of a Ribosome Profiling protocol to study gene expression in the thermotolerant yeast Kluyveromyces marxianus. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/478964v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1268da7org.highwire.dtl.DTLVardef@1ddff1org.highwire.dtl.DTLVardef@1597da4org.highwire.dtl.DTLVardef@47c2ae_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗