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Muscleblind-1 Interacts With Tubulin mRNAs to Regulate the Microtubule cytoskeleton in C. elegans Mechanosensory Neurons

Dharmendra Puri, Sunanda Sharma, Sarbani Samaddar, Sruthy Ravivarma, Sourav Banerjee, Anindya Ghosh-Roy

Abstract

Regulation of the microtubule cytoskeleton is crucial for the development and maintenance of neuronal architecture, and recent studies have highlighted the significance of regulated RNA processing in the establishment and maintenance of neural circuits. In a genetic screen conducted using mechanosensory neurons of C. elegans, we identified a mutation in muscleblind-1/mbl-1 as a suppressor of loss of kinesin-13 family microtubule destabilizing factor klp-7. Muscleblind-1(MBL-1) is an RNA-binding protein that regulates the splicing, localization, and stability of RNA. Our findings demonstrate that mbl-1 is required cell-autonomously for axon growth and proper synapse positioning in the posterior lateral microtubule (PLM) neuron. Loss of mbl-1 leads to increased microtubule dynamics and mixed orientation of microtubules in the anterior neurite of PLM.

Introduction

A highly ordered functional neuronal circuit comprises polarized nerve cells, which are compartmentalized into dendrites and axons for unidirectional reception and transmission of information. Several reports suggest that the structural and functional polarity of neurons relies on cytoskeletal elements within the neuron [14]. These cytoskeletal elements are regulated by intra- and extra-cellular signal transduction pathways during neuronal polarization [57]. The organization of the microtubule cytoskeleton in neurons plays a crucial role in directing neuronal polarization [8].

Materials and method

C. elegans genetics

C. elegans strains were cultured on standard Nematode Growth Medium (NGM) plates seeded with OP50 Escherichia coli bacteria at 20°C [86]. All the loss-of-function mutant alleles were denoted as “0”. For instance, the mbl-1(tm1563) mutant was represented as mbl-1(0). The N2 Bristol wild-type strain was used to eliminate background mutations, while CB4856 Hawaiian isolates were employed for restriction fragment length polymorphism (RFLP) mapping. The list of mutant strains and transgenic reporter strains used in this study can be found in S5 and S6 Tables, respectively. Additionally, the list of newly generated transgenes, carrying extrachromosomal arrays utilized in this study is included in S6 Table.

Results

Loss of muscleblind-1 (mbl-1) suppresses the ectopic neurite growth phenotype in touch neurons of klp-7(0) mutant

In C. elegans, six mechanosensory neurons are responsible for gentle touch sensation, including a pair of Anterior Lateral Microtubule (ALM) and a pair of Posterior Lateral Microtubule (PLM) neurons (white arrowheads, Fig 1A). ALM and PLM neurons extend their axons laterally towards the anterior side and establish connections with their respective postsynaptic neurons through a ventral synaptic branch (white arrows, Fig 1A). Additionally, the PLM neuron extends a short posterior neurite into the tail of the animal (double-sided white arrow, Fig 1A). Previous research has shown that loss of microtubule depolymerizing protein kinesin-13/KLP-7 leads to an ectopic extension of the posterior neurite from the ALM cell body (yellow arrow, Fig 1A) due to increased stabilization of the microtubule cytoskeleton [7]. 

Discussion

In this study, we uncovered a cell-autonomous role of the RNA-binding protein MBL-1 in neurite outgrowth and synapse formation in touch neurons (Fig 8H). Our findings demonstrated that microtubule stability in PLM touch neurons is compromised in mbl-1 mutants due to reduced levels of MEC-7 (β-tubulin) and MEC-12 (α-tubulin). Further investigation revealed that MBL-1 directly binds to the mec-7 and mec-12 transcripts regulating their stability (Fig 8H). Additionally, mbl-1 plays a crucial role in the proper positioning of synapses in PLM neurons by regulating sad-1 (Fig 8H).

Acknowledgments

We would like to thank the National Bio-Resource Project, Japan, and the Caenorhabditis Genetics Centre for strains. We thank Andrew Chisholm and Yishi Jin for their support and guidance at the initial stage of this project. The ju1128 mutant was isolated in their labs. We thank Neeraj Singh and Pankajam Thyagarajan for their assistance in WGS data analysis. We thank Sibaram Behera for helping with touch assay experiments and for the help in Bioinformatics analysis. We thank Arnab Mukhopadhyay, Sandhya Koushika, Michael Nonet, and Cori Bargmann for their help with strains. We are also grateful to Yuji Kohara for sharing mbl-1 cDNA.

Citation: Puri D, Sharma S, Samaddar S, Ravivarma S, Banerjee S, Ghosh-Roy A (2023) Muscleblind-1 interacts with tubulin mRNAs to regulate the microtubule cytoskeleton in C. elegans mechanosensory neurons. PLoS Genet 19(8): e1010885. https://doi.org/10.1371/journal.pgen.1010885

Editor: Massimo A. Hilliard, The University of Queensland, AUSTRALIA

Received: September 14, 2022; Accepted: July 26, 2023; Published: August 21, 2023

Copyright: © 2023 Puri et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting information files.

Funding: This work was supported by the National Brain Research Centre core fund from the Department of Biotechnology, The Wellcome Trust DBT India Alliance (IA/I/13/1/500874), and a grant from the Science and Engineering Research Board (SERB: CRG/2019/002194) to A.G.R. This work is also supported by a grant from the Science and Engineering Research Board (Grant # CRG/2018/00425) to S.B. The Caenorhabditis Genetics Centre is supported by the National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

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