Biology subjects

Krishnan, V. P.

Publications and source records attributed to Krishnan, V. P..

4 recordsLinked to original sources

bioRxiv2024

Prp16 enables efficient splicing of introns with diverse exonic consensus elements in the short-intron rich Cryptococcus neoformans transcriptome

The DEAH box splicing helicase Prp16 in budding yeast governs spliceosomal remodeling from the branching conformation (C complex) to the exon ligation conformation (C* complex). In this study, we examined the genome-wide functions of Prp16 in the short intron-rich genome of the basidiomycete yeast Cryptococcus neoformans. The presence of multiple introns per transcript with intronic features more similar to higher eukaryotes makes it a promising model to study spliceosomal splicing. Using a promoter-shutdown conditional Prp16 knockdown strain, we uncovered its genome-wide but substrate-specific roles in C. neoformans splicing. The splicing functions of Prp16 are dependent on its helicase motif I and motif II that are conserved motifs for helicase activity. A small subset of introns spliced independent of Prp16 activity, were investigated to discover that exonic sequences at the 5 splice site (5SS) and 3 splice site (3SS) with stronger affinity for U5 loop 1 as a common feature in these introns. Furthermore, short (60-100nts) and ultra-short introns (<60nts) prevalent in the C. neoformans transcriptome were more sensitive to Prp16 knockdown than longer introns, indicating Prp16 is required for the efficient splicing of short and ultra-short introns. We propose that stronger U5 snRNA-pre-mRNA interactions enable the efficient transition of the spliceosome from the first to the second catalytic confirmation in Prp16 knockdown, particularly for short introns and introns with suboptimal features. This study provides insights into the fine-tuning spliceosomal helicase functions with variations in cis-element features.

molecular biology↗

bioRxiv2024

Cardiac Outflow tract septation defects in a DiGeorge syndrome model respond to Minoxidil treatment.

BackgroundThe T-BOX transcription factor TBX1 is essential for the development of the pharyngeal apparatus and it is haploinsufficient in DiGeorge syndrome (DGS), a developmental anomaly associated with congenital heart disease and other abnormalities. The murine model recapitulates the heart phenotype and showed collagen accumulation. MethodsWe first used a cellular model to study gene expression during cardiogenic differentiation of WT and Tbx1-/- mouse embryonic stem cells. Then we used a mouse model of DGS to test whether interfering with collagen accumulation using an inhibitor of lysyl hydroxylase would modify the cardiac phenotype of the mutant. Results and conclusionsIn the cell differentiation model, loss of Tbx1 was associated with up regulation of a subset of ECM-related genes, including several collagen genes. In the in vivo model, early prenatal treatment with Minoxidil, a lysyl hydroxylase inhibitor, ameliorated the cardiac outflow tract septation phenotype in Tbx1 mutant fetuses, but it had no effect on septation in WT fetuses. We conclude that TBX1 suppresses a subset of ECM-related genes. The partial rescue of the septation phenotype through Minoxidil treatment suggests that inhibiting collagen cross-linking reduces the impact of the phenotypic consequences of Tbx1 mutation.

developmental biology↗

bioRxiv2023

Cryptococcus neoformans Slu7 ensures nuclear positioning during mitotic progression through RNA splicing

The position of the nucleus before it divides during mitosis is variable in different budding yeasts. Studies in the pathogenic intron-rich fungus Cryptococcus neoformans reveal that the nucleus moves entirely into the daughter bud before its division. Here, we report functions of a zinc finger motif containing spliceosome protein C. neoformans Slu7 (CnSlu7) in cell cycle progression. The budding yeast and fission yeast homologs of Slu7 have predominant roles for intron 3 splice site definition during pre-mRNA splicing. Using a conditional knockdown strategy, we show CnSlu7 is an essential factor for viability and is required for efficient cell cycle progression with major role during mitosis. Aberrant nuclear migration, including improper positioning of the nucleus as well as the spindle, were frequently observed in cells depleted of CnSlu7. However, cell cycle delays observed due to Slu7 depletion did not activate the Mad2-dependent spindle assembly checkpoint (SAC). Mining of the global transcriptome changes in the Slu7 knockdown strain identified downregulation of transcripts encoding several cell cycle regulators and cytoskeletal factors for nuclear migration, and the splicing of specific introns of these genes was CnSlu7 dependent. To test the importance of splicing activity of CnSlu7 on nuclear migration, we complemented Slu7 knockdown cells with an intron less PAC1 minigene and demonstrated that the nuclear migration defects were significantly rescued. These findings show that CnSlu7 regulates the functions of diverse cell cycle regulators and cytoskeletal components, ensuring timely cell cycle transitions and nuclear division during mitosis.

microbiology↗

bioRxiv2023

ENDOTHELIAL GENE REGULATORY ELEMENTS ASSOCIATED WITH CARDIOPHARYNGEAL LINEAGE DIFFERENTIATION

Endothelial cells (EC) differentiate from multiple sources, including the cardiopharyngeal mesoderm, which gives rise also to cardiac and branchiomeric muscles. Here, we used a cardiogenic mesoderm cell differentiation model that also activates an endothelial transcription program to identify endothelial regulatory elements activated in early cardiogenic mesoderm. Integrating our chromatin remodeling and gene expression data with available single-cell RNA-seq data from mouse embryos, we identified 101 putative regulatory elements of EC genes. We then applied a machine-learning strategy, trained on validated enhancers, to predict the probability of the sequences to function as enhancers. The computational assay determined that 50% of these sequences were likely enhancers, some of which have been previously reported. We also identified a smaller set of regulatory elements of well-known EC genes and validated them using genetic and epigenetic perturbation. Finally, we used the integration of multiple data sources and computational tools to search for transcriptional factor binding motifs. In conclusion, we identified novel EC regulatory sequences with a high likelihood to be enhancers, and we validated a subset of them using computational and cell culture models. Motif analyses revealed that the core EC transcription factors GATA/ETS/FOS is a likely driver of EC differentiation in cardiopharyngeal mesoderm.

molecular biology↗