Biology subjects

Kiniry, S. J.

Publications and source records attributed to Kiniry, S. J..

5 recordsLinked to original sources

bioRxiv2025

Translation of numerous upstream open reading frames rather than their products is essential for proliferation of human cells of distinct origin

Upstream open reading frames (uORFs) are a widespread class of translated regions (translons) occurring in 5' leaders of mRNAs and serving critical roles in post-transcriptional regulation. However, their specific biological activities in human cells remains to be fully elucidated. Here, we conducted a genome-wide CRISPR-Cas9 loss-of-function screen of 978 uORFs identified with ribosome profiling, across human cell lines of distinct origin (HAP1, A549 and HEK293T). A total of 155 uORFs were identified as being essential for cell proliferation. These uORFs showed a high cell-type specificity, with only a few being universally essential. Subsequent analysis has revealed that the primary reason underlying the uORF essentiality is not encoded micropeptides, but rather cis-regulatory mechanisms. Moreover, uORFs located within short 5' UTRs were disproportionately sensitive to frameshift-inducing indels, which frequently lead to the uORF extension and overlap with the coding region (CDS), resulting in translational repression. Finally, by intersecting regions of essential uORF with ClinVar and dbSNP datasets, we identified naturally occurring variants with the potential to disrupt their function and contribute to disease phenotypes. These findings highlight a pervasive and underappreciated layer of translational control in human cells and establish uORFs as critical cis-regulatory elements with potential relevance to human health.

molecular biology↗

bioRxiv2022

Thousands of human non-AUG extended proteoforms lack evidence of evolutionary selection among mammals

The synthesis of most proteins begins at AUG codons, yet a small number of non-AUG initiated proteoforms are also known. Here we used publicly available ribo-seq data with phylogenetic approaches to identify novel, previously uncharacterised non-AUG proteoforms. Unexpectedly we found that the number of non-AUG proteoforms identified with ribosome profiling data greatly exceeds those with strong phylogenetic support. We identified an association between proteoforms with alternative N-termini and multiple compartmentalisation of corresponding gene products. In dozens of genes N-terminal extensions encode localisation signals, including mitochondrial presequence and signal peptides. While the majority of non-AUG initiated proteoforms occur in addition to AUG initiated proteoforms, in few cases non-AUG appears to be the only start. This suggests that alternative compartmentalisation is not the only function of non-AUG initiation. Taking a conservative approach, we updated annotation of several genes in the latest GENCODE version in human and mouse where non-AUG initiated proteofoms are supported by both, ribosome profiling and phylogenetic evidence. Yet, the number of such extensions is likely much higher. Thousands of non-AUG proteoforms supported only by ribosome profiling suggest that they may evolve neutrally. Indeed, expression of some may not be consequential, i.e. when N-termini is processed or they have identical biochemical properties. Nonetheless they may contribute to immune response as antigen sources. It is also possible that some proteoforms accrued useful functions only recently and evolved under purifying selection in a narrow phylogenetic group. Thus, further characterisation is important for understanding their phenotypical and clinical significance.

genomics↗

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↗

bioRxiv2019

Selection shapes synonymous stop codon use in mammals

Phylogenetic models of the evolution of protein-coding sequences can provide insights into the selection pressures that have shaped them. In the application of these models synonymous nucleotide substitutions, which do not alter the encoded amino acid, are often assumed to have limited functional consequences and used as a proxy for the neutral rate of evolution. The ratio of nonsynonymous to synonymous substitution rates is then used to categorize the selective regime that applies to the protein (e.g. purifying selection, neutral evolution, diversifying selection). Here, we extend these models to explore the extent of purifying selection acting on substitutions between synonymous stop codons. Using a large collection of coding sequence alignments we estimate that a high proportion (approximately 57%) of mammalian genes are affected by selection acting on stop codon preference. This proportion varies substantially by codon, with UGA stop codons far more likely to be conserved. Genes with evidence of selection acting on synonymous stop codons have distinctive characteristics, compared to unconserved genes with the same stop codon, including enrichment for specific functional properties. Notably, genes with conserved stop codons have longer 3 UTRs and are associated with shorter mRNA half-life than other genes. The coding regions of these genes are also much more likely to be under strong purifying selection pressure. Our results suggest that the preference for UGA stop codons found in many multicellular eukaryotes is selective rather than mutational in origin.

evolutionary biology↗