Biology subjects

Das, R. M.

Publications and source records attributed to Das, R. M..

3 recordsLinked to original sources

bioRxiv2024

Golgi compaction facilitates microtubule nucleation to drive adult vertebrate peripheral neuron regeneration

Peripheral neurons regenerate in response to injury, but their cell intrinsic processes are poorly understood and seldom sufficient to effect clinical restoration. Using a novel assay for high-resolution live imaging of regenerating adult vertebrate neurons, we identify acute fragmentation and rapid recompaction of the somatic Golgi as a key driver of peripheral neuron regeneration, implicating this organelle as a therapeutic target in an area of clinical unmet need. Compaction of the fragmented Golgi facilitates stepwise recruitment of the microtubule nucleation factors AKAP9 and {gamma}-tubulin within a discrete period that corresponds with acentrosomal Golgi-mediated microtubule nucleation. Furthermore, disruption of AKAP9 or {gamma}-tubulin recruitment compromises microtubule nucleation leading to impaired regeneration. Crucially, these mechanisms are conserved in the contexts of in vivo rat sciatic nerve transection and in primary human peripheral neurons. This work transforms our understanding of the cell intrinsic mechanisms that render injured peripheral neurons competent to initiate axon regeneration.

cell biology↗

bioRxiv2022

Computationally-guided design and selection of ribosomal active site mutants with high activity

Understanding how modifications to the ribosome affect function has implications for studying ribosome biogenesis, building minimal cells, and repurposing ribosomes for synthetic biology. However, efforts to design sequence-modified ribosomes have been limited because point mutations in the ribosomal RNA (rRNA), especially in the catalytic active site (peptidyl transferase center; PTC), are often functionally detrimental. Moreover, methods for directed evolution of rRNA are constrained by practical considerations (e.g., library size). Here, to address these limitations, we developed a computational rRNA design approach for screening guided libraries of mutant ribosomes. Our method includes in silico library design and selection using a Rosetta stepwise Monte Carlo method (SWM), library construction and in vitro testing, and functional characterization in vivo. As a model, we apply our method to making modified ribosomes with mutant PTCs. We engineer ribosomes with as many as 30 mutations in their PTCs, highlighting previously unidentified epistatic interactions, and show that SWM helps identify sequences with beneficial phenotypes as compared to random library sequences. We further demonstrate that some variants improve cell growth in vivo, relative to wild type ribosomes. We anticipate that SWM design may serve as a powerful tool for high-resolution rRNA design.

synthetic biology↗

bioRxiv2020

RNA 3D modeling with FARFAR2, online

Understanding the three-dimensional structure of an RNA molecule is often essential to understanding its function. Sampling algorithms and energy functions for RNA structure prediction are improving, due to the increasing diversity of structural data available for training statistical potentials and testing structural data, along with a steady supply of blind challenges through the RNA Puzzles initiative. The recent FARFAR2 algorithm enables near-native structure predictions on fairly complex RNA structures, including automated selection of final candidate models and estimation of model accuracy. Here, we describe the use of a publicly available webserver for RNA modeling for realistic scenarios using FARFAR2, available at https://rosie.rosettacommons.org/farfar2. We walk through two cases in some detail: a simple model pseudoknot from the frameshifting element of beet western yellows virus modeled using the "basic interface" to the webserver, and a replication of RNA-Puzzle 20, a metagenomic twister sister ribozyme, using the "advanced interface." We also describe example runs of FARFAR2 modeling including two kinds of experimental data: a c-di-GMP riboswitch modeled with low resolution restraints from MOHCA-seq experiments and a tandem GA motif modeled with 1H NMR chemical shifts.

biochemistry↗