Biology subjects

Topkar, V. V.

Publications and source records attributed to Topkar, V. V..

4 recordsLinked to original sources

bioRxiv2025

RNA switch model for localization and translation of the myelin basic protein mRNA

Oligodendrocytes myelinate the central nervous system by extending cellular projections that ensheath axons and elongate to form lipid-rich myelin. Classic studies visualizing RNA dynamics showed that myelin basic protein (MBP), one of the most abundant myelin proteins, is locally synthesized at the myelin sheath through the transport and local translation of Mbp mRNA. Mbp transport requires its 1.5-kb 3 untranslated region (3 UTR) and prior work identified candidate sub-sequences that may act as cis-acting transport stimulating RNA elements, including one with putative secondary structure. Here, a high-throughput reporter assay, dimethyl sulfate (DMS)-based RNA structure probing, and microscopy in primary rat oligodendrocytes identify a structured 127-nt region that we name the Mbp localization signal (MLS) as both necessary and sufficient for RNA enrichment to oligodendrocyte projections. Lysate pulldown experiments further identify hnRNP-F - a known constituent of the Mbp RNA granule that can suppress mRNA translation - as associated with the MLS; paradoxically, binding of this protein should compete with the ordered MLS RNA structure. These results suggest a model in which the MLS switches between two RNA conformations with distinct protein partners during the transition from Mbp mRNA transport to Mbp translation at the myelin sheath. Such regulation of RNA behavior by structure switching may generalize to other eukaryotic mRNAs whose behaviors shift across space and time. Significance StatementIn the brain, oligodendrocyte cells generate myelin, a type of insulation that wraps around neuronal axons in order to facilitate fast electrical signaling. A critical step in myelination is the local translation of MBP (myelin basic protein) in the myelin sheath. This requires the transport of Mbp mRNA, an incompletely understood phenomenon that we revisit using two recent approaches for mRNA structure and function. We refine a 127-nt region that is necessary and sufficient for mRNA transport to the myelin sheath. A proteomic screen reveals that this myelin localization signal (MLS) associates with a translation-suppressing protein called hnRNP-F, suggesting a model where Mbp mRNA switches between two states, one for transport and one for translation at the myelin sheath.

cell biology↗

bioRxiv2021

Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics

Therapeutic mRNAs and vaccines are being developed for a broad range of human diseases, including COVID-19. However, their optimization is hindered by mRNA instability and inefficient protein expression. Here, we describe design principles that overcome these barriers. We develop a new RNA sequencing-based platform called PERSIST-seq to systematically delineate in-cell mRNA stability, ribosome load, as well as in-solution stability of a library of diverse mRNAs. We find that, surprisingly, in-cell stability is a greater driver of protein output than high ribosome load. We further introduce a method called In-line-seq, applied to thousands of diverse RNAs, that reveals sequence and structure-based rules for mitigating hydrolytic degradation. Our findings show that "superfolder" mRNAs can be designed to improve both stability and expression that are further enhanced through pseudouridine nucleoside modification. Together, our study demonstrates simultaneous improvement of mRNA stability and protein expression and provides a computational-experimental platform for the enhancement of mRNA medicines.

molecular biology↗

bioRxiv2020

Anomalous reverse transcription through chemical modifications in polyadenosine stretches

Thermostable reverse transcriptases are workhorse enzymes underlying nearly all modern techniques for RNA structure mapping and for transcriptome-wide discovery of RNA chemical modifications. Despite their wide use, these enzymes behaviors at chemical modified nucleotides remain poorly understood. Wellington-Oguri et al. recently reported an apparent loss of chemical modification within putatively unstructured polyadenosine stretches modified by dimethyl sulfate or 2 hydroxyl acylation, as probed by reverse transcription. Here, re-analysis of these and other publicly available data, capillary electrophoresis experiments on chemically modified RNAs, and nuclear magnetic resonance spectroscopy on A12 and variants show that this effect is unlikely to arise from an unusual structure of polyadenosine. Instead, tests of different reverse transcriptases on chemically modified RNAs and molecules synthesized with single 1-methyladenosines implicate a previously uncharacterized reverse transcriptase behavior: near-quantitative bypass through chemical modifications within polyadenosine stretches. All tested natural and engineered reverse transcriptases (MMLV; SuperScript II, III, and IV; TGIRT-III; and MarathonRT) exhibit this anomalous bypass behavior. Accurate DMS-guided structure modeling of the polyadenylated HIV-1 3 untranslated region RNA requires taking into account this anomaly. Our results suggest that poly(rA-dT) hybrid duplexes can trigger unexpectedly effective reverse transcriptase bypass and that chemical modifications in poly(A) mRNA tails may be generally undercounted.

biochemistry↗

bioRxiv2019

Ribosolve: Rapid determination of three-dimensional RNA-only structures

The discovery and design of biologically important RNA molecules is dramatically outpacing three-dimensional structural characterization. To address this challenge, we present Ribosolve, a hybrid method integrating moderate-resolution cryo-EM maps, chemical mapping, and Rosetta computational modeling, and demonstrate its application to thirteen previously unknown 119-to 338-nucleotide protein-free RNA-only structures: full-length Tetrahymena ribozyme, hc16 ligase with and without substrate, full-length V. cholerae and F. nucleatum glycine riboswitch aptamers with and without glycine, Mycobacterium SAM-IV riboswitch with and without S-adenosylmethionine, and computer-designed spinach-TTR-3, eterna3D-JR_1, and ATP-TTR-3 with and without AMP. Blind challenges, prospective compensatory mutagenesis, internal controls, and simulation benchmarks validate the Ribosolve models and establish that modeling convergence is quantitatively predictive of model accuracy. These results demonstrate that RNA-only 3D structure determination can be rapid and routine.

biophysics↗