Biology subjects

Kryvoruchko, I. S.

Publications and source records attributed to Kryvoruchko, I. S..

6 recordsLinked to original sources

bioRxiv2026

Loss-of-function phenomics, ncORFs, and ambiguity of mutant phenotypes in Medicago truncatula

Non-canonical open reading frames (ncORFs) are an emerging area of research that is quickly gaining momentum. Many peptides and proteins missed in initial annotation efforts (ncProts) were subsequently shown to be crucial for a wide range of biological processes. The discovery of ncORFs continues to improve the accuracy of loss-of-function studies because they often occupy the same genomic spaces as annotated ORFs. While databases of mutant phenotypes linked to genomic loci exist in a few species, none of these databases integrate the information on ncORFs present in already characterized loci. In this study, we introduce a nearly comprehensive loss-of-function phenomics dataset of Medicago truncatula (673 loci characterized over the past 30 years), which was integrated as a new track into the genome browser of this organism. This dataset helped critically analyze the potential contribution of ncORFs to published phenotypes. We detected mass spectrometry (MS)-validated ncORFs in 10 characterized genes, including major regulators of development and symbiotic relationships. We also found conserved ncORFs in 113 characterized genes, including four genes with highly conserved ncORFs. In some studies, the contribution of these ncORFs can be ruled out, while in others it cannot. Using real examples, we systematized ambiguities associated with ncORFs. Furthermore, we highlighted little-known trans effects of insertional mutagenesis on splicing as contributors to that ambiguity. Finally, our meta-analysis of published phenotypes revealed that different protein classes have significantly different (unique) proportions of unconditional, conditional, and neutral phenotypes, potentially reflecting their relative functional importance. Significance statementThis study is the first to merge a nearly comprehensive inventory of loss-of-function studies in a eukaryotic organism with the information on novel MS-validated and conserved ncORFs.

genetics↗

bioRxiv2025

ShiftSCAN, a program that predicts potential alternative sources of mass spectrometry-derived peptides, improves the accuracy of studies on novel amino acid sequences

Mass spectrometry (MS) proteomics is currently the most powerful tool for identifying both annotated proteins and proteins translated from non-canonical open reading frames or unusual genetic events. With this method, numerous novel protein-coding loci have been discovered by searching for short fragments of hypothetical longer peptides and polypeptides. Apart from the validation of translation from mRNA transcripts, MS proteomics has been instrumental for the detection of peptides encoded by non-mRNA transcripts. A special application field of MS proteomics is studies on programmed ribosomal frameshifting (PRF), where the detection of chimeric peptides produced from two different reading frames is vital. Each novel chimeric peptide is thought to originate from a certain genetic locus. However, due to the short length of MS peptides, there is a possibility that MS-supported chimeric peptides are produced by additional (alternative) loci via PRF. This scenario evaded due attention because the contribution of non-canonical peptides and proteins to the functional diversity of proteomes is still thought to be minor. Recent studies have challenged this paradigm. To the best of our knowledge, our group was the first to include alternative chimeric sources into the analysis pipeline. This resulted in a much higher certainty about loci responsible for the production of unusual peptides. This certainty is crucial for the functional characterization of such loci. At the same time, our study revealed enormous diversity of potential alternative sources for a subset of MS-supported non-canonical peptides. Here, we present a highly flexible program that predicts alternative chimeric and non-chimeric sources of peptides detected by MS proteomics.

bioinformatics↗

bioRxiv2025

A universal pipeline MosaicProt enables large-scale modeling and detection of chimeric protein sequences for studies on programmed ribosomal frameshifting

Peptides and proteins produced by programmed ribosomal frameshifting (PRF) are well-known in viruses. In non-viral systems, only a few examples of such chimeric sequences have been documented until recently. Two new studies, one in humans and one in plants, showed that chimeric peptides are numerous and diverse. In humans, their discovery was possible due to focusing on sequences with naturally repeated codons. This way, many candidate sequences with mass spectrometry (MS) proteomics-based support for translation have been identified. In the plant study, our group discovered MS-validated chimeric peptides using a unique modeling algorithm, which is described and made available here. Our pipeline enables the identification of chimeric peptides in any organism for which transcript sequences and MS proteomic data are available. By design, our approach does not require prior knowledge about sequence similarity to already characterized PRF sites and can detect forward and backward frameshifts by 1 and 2 nucleotides. Thus, our pipeline opens a path for uncovering previously unknown PRF events across various transcript types, potentially broadening our understanding of proteome diversity.

bioinformatics↗

bioRxiv2024

Discovery of diverse chimeric peptides in a eukaryotic proteome sets the stage for the experimental proof of the mosaic translation hypothesis

The high complexity of eukaryotic organisms enabled their evolutionary success, which became possible due to the diversification of eukaryotic proteomes. Various mechanisms contributed to this process. Alternative splicing had the largest known impact among these mechanisms: tens or hundreds of protein isoforms produced from a single genetic locus. Earlier, we hypothesized that along with alternative splicing, a different but conceptually similar mechanism creates novel versions of existing proteins in all eukaryotes. However, this mechanism acts at the level of translation, where the novelty of an amino acid sequence is achieved via multiple programmed ribosomal frameshifting. This mechanism, which is termed mosaic translation, is very difficult to demonstrate even with the most up-to-date molecular tools. Thus, it remained unnoticed so far. Using only a portion of all mass spectrometry proteomic data generated from various organs of the model plant Medicago truncatula, we attempted the first step toward the experimental proof of this hypothesis. Our original in silico approach resulted in the discovery of two candidates for mosaic proteins (homologs of EF1 and RuBisCo) and 154 candidates for chimeric peptides. Chimeric peptides and polypeptides are produced in the course of one ribosomal frameshifting event and may correspond to parts of mosaic proteins. In addition, our analysis reveals the possibility of translation of chimeric peptides from five ribosomal RNA transcripts, ten long non-coding RNA transcripts, and one transfer RNA transcript. These findings are very novel and will be the basis for experimental validation in future studies. In this work, we present multiple lines of indirect evidence that support the validity of our in silico data.

biochemistry↗

bioRxiv2020

GmVTL1 is an iron transporter on the symbiosome membrane of soybean with an important role in nitrogen fixation.

O_LILegumes establish symbiotic relationships with soil bacteria (rhizobia), housed in nodules on plant roots. The plant supplies carbon substrates and other nutrients to the bacteria in exchange for fixed nitrogen. The exchange occurs across a plant-derived symbiosome membrane (SM), which encloses rhizobia to form a symbiosome. Iron supplied by the plant is crucial for the rhizobial enzyme nitrogenase that catalyses N2 fixation, but the SM iron transporter has not been identified. C_LIO_LIWe use complementation of yeast and plant mutants, real-time PCR, hairy root transformation, microscopy and proteomics to demonstrate the role of soybean GmVTL1 and 2. C_LIO_LIBoth are members of the vacuolar iron transporter family and homologous to Lotus japonicus SEN1 (LjSEN1), previously shown to be essential for N2 fixation. GmVTL1 expression is enhanced in nodule infected cells and both proteins are localised to the SM. C_LIO_LIGmVTL1 and 2 transport iron in yeast and GmVTL1 restores N2 fixation when expressed in the Ljsen1 mutant. C_LIO_LIThree GmVTL1 amino acid substitutions that reduce iron transport in yeast also block N2 fixation in Ljsen1 plants. C_LIO_LIWe conclude GmVTL1 is responsible for transport of iron across the SM to bacteroids and plays a crucial role in the N2-fixing symbiosis. C_LI

plant biology↗

bioRxiv2017

Medicago truncatula copper transporter1 (MtCOPT1) delivers copper for symbiotic nitrogen fixation

* Copper is an essential nutrient for symbiotic nitrogen fixation. This element is delivered by the host plant to the nodule, where membrane copper transporter would introduce it into the cell to synthesize cupro-proteins.\n\n* COPT family members in model legume Medicago truncatula were identified and their expression determined. Yeast complementation assays, confocal microscopy, and phenotypical characterization of a Tnt1 insertional mutant line were carried out in the nodule-specific M.truncatula COPT family member.\n\n* Medicago truncatula genome encodes eight COPT transporters. MtCOPT1 (Medtr4g019870) is the only nodule-specific COPT gene. It is located in the plasma membrane of the differentiation, interzone and early fixation zones. Loss of MtCOPT1 function results in a copper-mitigated reduction of biomass production when the plant obtains its nitrogen exclusively from symbiotic nitrogen fixation. Mutation of MtCOPT1 results in diminished nitrogenase activity in nodules, likely an indirect effect from the loss of a copper-dependent function, such as cytochrome oxidase activity in copt1-1 bacteroids.\n\n* These data are consistent with a model in which MtCOPT1 transports copper from the apoplast into nodule cells to provide copper for essential metabolic processes associated with symbiotic nitrogen fixation.

plant biology↗