Biology subjects

Kufel, J.

Publications and source records attributed to Kufel, J..

6 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↗

bioRxiv2025

The Arabidopsis deNADding enzyme DXO1 modulates the plant immunity response.

O_LIDXO1, the only DXO homolog in Arabidopsis, due to its plant-specific features, has a strong deNADding enzymatic activity but no apparent role in 5'QC. Most molecular and morphological changes observed so far in dxo1 mutant plants depended on the plant-specific N-terminal domain of the protein. C_LIO_LIOur investigation revealed the importance of DXO1 enzymatic activity in the plant immune response. We observed that dxo1-2 knockout mutant and transgenic dxo1-2 lines expressing a DXO1 variant either catalytically inactive or lacking the N-terminal domain exhibited enhanced resistance to Pseudomonas syringae, accompanied by marked changes in the expression of key pathogenesis markers. Other markers of plant immunity, such as callose deposition and production of ROS, were strongly induced by elf18 and flg22. C_LIO_LIOur results strongly suggest that both DXO1 features, the N-terminal domain and its catalytic site, contribute to regulating plant immunity. This is the first observation revealing the involvement of the DXO1 enzymatic activity in plant physiology. C_LIO_LIMoreover, our analyses showed that dxo1-2 mutation altered the expression of a large group of defense-related genes, affected their mRNAs stability, and delayed activation of MAP kinases. Therefore, we postulate that DXO1 protein deregulates defense against Pst infection at both the transcriptional and post-transcriptional level. C_LI

molecular biology↗

bioRxiv2023

Ribosome Decision Graphs for the Representation of Eukaryotic RNA Translation Complexity

The application of ribosome profiling has revealed an unexpected abundance of translation in addition to that responsible for the synthesis of previously annotated protein-coding regions. Multiple short sequences have been found to be translated within single RNA molecules, both within annotated protein-coding and non-coding regions. The biological significance of this translation is a matter of intensive investigation. However, current schematic or annotation-based representations of mRNA translation generally do not account for the apparent multitude of translated regions within the same molecules. They also do not take into account the stochasticity of the process that allows alternative translations of the same RNA molecules by different ribosomes. There is a need for formal representations of mRNA complexity that would enable the analysis of quantitative information on translation and more accurate models for predicting the phenotypic effects of genetic variants affecting translation. To address this, we developed a conceptually novel abstraction that we term Ribosome Decision Graphs (RDGs). RDGs represent translation as multiple ribosome paths through untranslated and translated mRNA segments. We termed the later translons. Non-deterministic events, such as initiation, re-initiation, selenocysteine insertion or ribosomal frameshifting are then represented as branching points. This representation allows for an adequate representation of eukaryotic translation complexity and focuses on locations critical for translation regulation. We show how RDGs can be used for depicting translated regions, analysis of genetic variation and quantitative genome-wide data on translation for characterisation of regulatory modulators of translation.

genomics↗

bioRxiv2022

Arabidopsis DXO1 affects the processing of precursors of cytoplasmic and chloroplast ribosomal RNA.

Decapping 5-3 exoribonucleases from the DXO/Rai1 family, are highly conserved among eukaryotes and exhibit diverse enzymatic activities depending on the organism. The biochemical and structural properties of the plant DXO1 differ from the yeast and animal counterparts, which is reflected in the in vivo functions of this enzyme. Here we show that Arabidopsis DXO1 contributes to the efficient processing of rRNA precursors in both nucleolar/cytosol and chloroplast maturation pathways. However, processing defects in DXO1-deficient plants do not depend on the catalytic activity of the enzyme but rely on its plant-specific N-terminal extension. Our RNA sequencing analyses show that the dxo1 mutation deregulates the expression of many ribosomal protein genes, most likely leading to inefficient or delayed pre-rRNA maturation. Strikingly, some of the observed molecular and morphological phenotypes of dxo1 plants are suppressed by the knock-down of XRN3, providing evidence for functional interaction between DXO1 and XRN proteins. HIGHLIGHTArabidopsis DXO1 protein regulates the expression of genes encoding ribosomal proteins and contributes to the correct processing of ribosomal RNA precursors.

molecular biology↗

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↗

bioRxiv2021

Arabidopsis spliceosome factor SmD3 modulates immunity to Pseudomonas syringae infection

SmD3 is a core component of the small nuclear ribonucleoprotein (snRNP) that is essential for pre-mRNA splicing. The role of Arabidopsis SmD3 in plant immunity was assessed by testing sensitivity of smd3a and smd3b mutants to Pseudomonas syringae pv. tomato (Pst) DC3000 infection and its pathogenesis effectors flagellin (flg22), EF-Tu (elf18) and coronatine (COR). Both smd3 mutants exhibited enhanced susceptibility to Pst accompanied by marked changes in the expression of key pathogenesis markers. mRNA levels of these factors were also altered upon treatment with Pseudomonas effectors. We showed that SmD3-b dysfunction impairs mainly stomatal immunity as a result of defects in stomatal development. Our genome-wide transcriptome analysis of the smd3b-1 mutant infected with Pst revealed that lack of SmD3-b deregulates defense against Pst infection at the transcriptional and posttranscriptional levels including defects in splicing and an altered pattern of alternative splicing. Other changes in the smd3b-1 mutant involved enhanced elf18- and flg22-induced callose deposition, reduction of flg22-triggered production of early ROS and boost of secondary ROS caused by Pst infection. Together, our data indicate that SmD3 contributes to the plant immune response possibly via regulation of mRNA splicing of key pathogenesis factors.

molecular biology↗