Biology subjects

Ramakrishna, M.

Publications and source records attributed to Ramakrishna, M..

3 recordsLinked to original sources

bioRxiv2018

Biotinylation by proximity labelling favours unfolded proteins

Folded enzymes are essential for life, but there is limited in vivo information about how locally unfolded protein regions contribute to biological functions. Intrinsically Disordered Regions (IDRs) are enriched in disease-linked and multiply post-translationally modified proteins. The extent of foldability of predicted IDRs is difficult to measure due to significant technical challenges to survey in vivo protein conformations on a proteome-wide scale. We reasoned that IDRs should be more accessible to targeted in vivo biotinylation than more ordered protein regions, if they retain their flexibility in vivo. Indeed, we observed a positive correlation of predicted IDRs and biotinylation density across four independent large-scale proximity proteomics studies that together report >20 000 biotinylation sites. We show that biotin painting is a promising approach to fill gaps in knowledge between static in vitro protein structures, in silico disorder predictions and in vivo condition-dependent subcellular plasticity using the 80S ribosome as an example.

cell biology↗

bioRxiv2018

Unbiased dynamic characterization of RNA-protein interactions by OOPS

Current methods for the identification of RNA-protein interactions require a quantity and quality of sample that hinders their application, especially for dynamic biological systems or when sample material is limiting. Here, we present a new approach to enrich RNA-Binding Proteins (RBPs): Orthogonal Organic Phase Separation (OOPS), which is compatible with downstream proteomics and RNA sequencing. OOPS enables recovery of RBPs and free protein, or protein-bound RNA and free RNA, from a single sample in an unbiased manner. By applying OOPS to human cell lines, we extract the majority of known RBPs, and importantly identify additional novel RBPs, including those from previously under-represented cellular compartments. The high yield and unbiased nature of OOPS facilitates its application in both dynamic and inaccessible systems. Thus, we have identified changes in RNA-protein interactions in mammalian cells following nocodazole cell-cycle arrest, and defined the first bacterial RNA-interactome. Overall, OOPS provides an easy-to-use and flexible technique that opens new opportunities to characterize RNA-protein interactions and explore their dynamic behaviour.

cell biology↗

bioRxiv2018

Partially methylated domains are hypervariable in breast cancer and fuel widespread CpG island hypermethylation

Global loss of DNA methylation and CpG island (CGI) hypermethylation are regarded as key epigenomic aberrations in cancer. Global loss manifests itself in partially methylated domains (PMDs) which can extend up to megabases. However, the distribution of PMDs within and between tumor types, and their effects on key functional genomic elements including CGIs are poorly defined. Using whole genome bisulfite sequencing (WGBS) of breast cancers, we comprehensively show that loss of methylation in PMDs occurs in a large fraction of the genome and represents the prime source of variation in DNA methylation. PMDs are hypervariable in methylation level, size and distribution, and display elevated mutation rates. They impose intermediate DNA methylation levels incognizant of functional genomic elements including CGIs, underpinning a CGI methylator phenotype (CIMP). However, significant repression effects on cancer-genes are negligible as tumor suppressor genes are generally excluded from PMDs. The genomic distribution of PMDs reports tissue-of-origin of different cancers and may represent tissue-specific silent regions of the genome, which tolerate instability at the epigenetic, transcriptomic and genetic level.

cancer biology↗