Biology subjects

CHAKRABORTY, A.

Publications and source records attributed to CHAKRABORTY, A..

3 recordsLinked to original sources

bioRxiv2023

Intronic polyadenylation isoforms in the 5' part of genes constitute a source of microproteins and are involved in cell response to cisplatin

Transcript isoforms generated by intronic polyadenylation (IPA) are widely regulated in various biological processes and often encode protein isoforms. Microproteins are small proteins translated from small open reading frames (sORFs) in noncoding RNAs and mRNAs, but their production by IPA isoforms is unknown. Using 3-seq and long-read RNA-seq analyses in lung cancer cells, we show that cisplatin, a DNA-crosslinking anticancer agent, upregulates IPA isoforms relative to full-length mRNAs in long genes. A subset of cisplatin-regulated IPA isoforms are poorly associated with heavy polysomes and terminate upstream of the annotated translation initiation codon of genes. Such IPA isoforms in the PHF20 and PRKAR1B genes are associated with light polysomes, contain Ribo-Seq-supported sORFs in an alternative last exon within the annotated 5UTR part of genes, and are translated into microproteins. For PRKAR1B, the microprotein was detected by Western blot and immunofluorescence after transfection of a tagged isoform; and siRNA depletion of the endogenous IPA isoform, CRISPR deletion of the IPA site, or CRISPR mutation of the sORF initiation codon led to increased cell survival to cisplatin. Based on Ribo-Seq and mass-spectrometry data sets, we identified 156 genes producing both a canonical protein-coding mRNA and a microprotein-coding 5UTR-located IPA isoform (coined miP-5UTR-IPA isoform) regulated by cisplatin. Finally, the regulation of (miP-5UTR-)IPA versus full-length isoforms by cisplatin involved an inhibition of transcription processivity in a FANCD2 and senataxin-dependent manner. Altogether, these findings reveal the novel paradigm of miP-5UTR-IPA genes and their role in cancer cell response to a genotoxic agent. HIGHLIGHTS- Cisplatin increases intronic-polyadenylation versus full-length transcript isoforms in long genes through a FANCD2 and senataxin-dependent decrease of transcription processivity - A subset of cisplatin-regulated intronic-polyadenylation isoforms terminate in the annotated 5UTR part of genes and encode microproteins, thus we coined them miP-5UTR-IPA isoforms - The miP-5UTR-IPA isoform of PRKAR1B impacts cisplatin sensitivity and its effect is mediated by its small ORF - We identify 156 genes producing both a canonical protein-coding mRNA and a microprotein-coding miP-5UTR-IPA transcript

molecular biology↗

bioRxiv2023

A glycolytic metabolite restores DNA repair activity of polynucleotide kinase 3-phosphatase in polyglutamine (PolyQ) diseases

Huntingtons disease (HD) and spinocerebellar ataxia type 3 (SCA3) are the two most prevalent polyglutamine (polyQ) neurodegenerative diseases, caused by CAG (encoding glutamine) repeat expansion in the coding region of the huntingtin (HTT) and ataxin-3 (ATXN3) proteins, respectively. We have earlier reported that the activity, but not the protein level, of an essential DNA repair enzyme, polynucleotide kinase 3-phosphatase (PNKP), is severely abrogated in both HD and SCA3 resulting in accumulation of double-strand breaks in patients brain genome. While investigating the mechanistic basis for the loss of PNKP activity and accumulation of DNA double-strand breaks leading to neuronal death, we observed that PNKP interacts with the nuclear isoform of 6-phosphofructo-2-kinase fructose-2,6-bisphosphatase 3 (PFKFB3). Depletion of PFKFB3 markedly abrogates PNKP activity without changing its protein level. Notably, the levels of both PFKFB3 and its product fructose-2,6 bisphosphate (F2,6BP), an allosteric modulator of glycolysis, are significantly lower in the nuclear extracts of post-mortem brain tissues of HD and SCA3 patients. Supplementation of F2,6BP restored PNKP activity in the nuclear extracts of patients brain. Moreover, intracellular delivery of F2,6BP restored both the activity of PNKP and the integrity of transcribed genome in neuronal cells derived from striatum of HD mouse. Importantly, supplementing F2,6BP rescued the HD phenotype in Drosophila, suggesting F2,6BP to serve in vivo as a cofactor for the proper functionality of PNKP and thereby, of brain health. Our results thus provide a compelling rationale for exploring the therapeutic use of F2,6BP and structurally related compounds for treating polyQ diseases. SignificanceTo unravel the biological basis for the loss of PNKP activity in HD and SCA3, the two most prevalent polyglutamine neurodegenerative disorders, we analyzed PNKP interactome and found that the nuclear isoform of a glycolytic enzyme PFKFB3 associated with PNKP and other repair proteins forming a multiprotein complex. Surprisingly, we found that PFKFB3 and its biosynthetic product, F2,6BP are significantly low in the affected region of patients brain. Exogenous addition of F2,6BP restored PNKP activity in patients brain nuclear extract. Moreover, supplementing F2,6BP in HD cells and fruit flies restored genome integrity and rescued the disease symptoms. While there is no curative therapy for HD/SCA3, except symptom management, our discovery suggests that F2,6BP supplementation would be a promising therapeutic option.

biochemistry↗

bioRxiv2021

Prefusion conformation of SARS-CoV-2 receptor-binding domain favors interactions with human receptor ACE2

A new coronavirus pandemic COVID-19, caused by Severe Acute Respiratory Syndrome coronavirus (SARS-CoV-2), poses a serious threat across continents, leading the World Health Organization to declare a Public Health Emergency of International Concern. In order to block the entry of the virus into human host cells, major therapeutic and vaccine design efforts are now targeting the interactions between the SARS-CoV-2 spike (S) glycoprotein and the human cellular membrane receptor angiotensin-converting enzyme, hACE2. By analyzing cryo-EM structures of SARS-CoV-2 and SARS-CoV-1, we report here that the homotrimer SARS-CoV-2 S receptor-binding domain (RBD) that binds with hACE2 has expanded in size, undergoing a large conformational change relative to SARS-CoV-1 S protein. Protomer with the up-conformational form of RBD, which binds with hACE2, exhibits higher intermolecular interactions at the RBD-ACE2 interface, with differential distributions and the inclusion of specific H-bonds in the CoV-2 complex. Further interface analysis has shown that interfacial water promotes and stabilizes the formation of CoV-2/hACE2 complex. This interaction has caused a significant structural rigidification, favoring proteolytic processing of S protein for the fusion of the viral and cellular membrane. Moreover, conformational dynamics simulations of RBD motions in SARS-CoV-2 and SARS-CoV-1 point to the role in modification in the RBD dynamics and their likely impact on infectivity.

bioinformatics↗