O'Connor, P. B. | bioRxiv Science

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↗

bioRxiv2020

Unprecedentedly efficient CUG initiation of an overlapping reading frame in POLG mRNA yields novel protein POLGARF

While near cognate codons are frequently used for translation initiation in eukaryotes, their efficiencies are usually low (<10% compared to an AUG in optimal context). Here we describe a rare case of highly efficient near cognate initiation. A CUG triplet located in the 5 leader of POLG mRNA initiates almost as efficiently (~60-70%) as an AUG in optimal context. This CUG directs translation of a conserved 260 triplet-long overlapping ORF, which we call POLGARF (POLG Alternative Reading Frame). Translation of a short upstream ORF 5 of this CUG governs the ratio between DNA polymerase and POLGARF produced from a single POLG mRNA. Functional investigation of POLGARF points to extracellular signalling. While unprocessed POLGARF resides in the nucleoli together with its interacting partner C1QBP, serum stimulation results in rapid secretion of POLGARF C-terminal fragment. Phylogenetic analysis shows that POLGARF evolved ~160 million years ago due to an MIR transposition into the 5 leader sequence of the mammalian POLG gene which became fixed in placental mammals. The discovery of POLGARF unveils a previously undescribed mechanism of de novo protein-coding gene evolution. Significance StatementIn this study, we describe previously unknown mechanism of de novo protein-coding gene evolution. We show that the POLG gene, which encodes the catalytic subunit of mitochondrial DNA polymerase, is in fact a dual coding gene. Ribosome profiling, phylogenetic conservation, and reporter construct analyses all demonstrate that POLG mRNA possesses a conserved CUG codon which serves as a start of translation for an exceptionally long overlapping open reading frame (260 codons in human) present in all placental mammals. We called the protein encoded in this alternative reading frame POLGARF. We provide evidence that the evolution of POLGARF was incepted upon insertion of an MIR transposable element of the SINE family.

molecular biology↗