The RNA binding protein HNRNPA2B1 regulates RNA abundance and motor protein activity in neurites

Joelle Lo, Katherine F. Vaeth, Gurprit Bhardwaj, Neelanjan Mukherjee, Holger A. Russ, Jeffrey K. Moore, J. Matthew Taliaferro

Posted on: 24 September 2024

Preprint posted on 26 August 2024

Lo and colleagues explore the role of RNA-binding protein HNRNPA2B1 in regulating mRNA and motor protein function in neurons. Loss of HNRNPA2B1 leads to an accumulation of RNAs in neurites, disrupting motor protein activity.

Selected by Felipe Del Valle Batalla

Categories: cell biology, molecular biology, neuroscience

Graphical abstract provided by the authors describing the main findings of this work.

Background

RNA localization and local protein synthesis are crucial for the spatial and temporal regulation of gene expression, especially in neurons, where they play an essential role in neurite development and function (Das, Vera, Gandin, Singer, & Tutucci, 2021; Lin, van Tartwijk, & Holt, 2021).Localized RNA and protein synthesis is, in part, supported by organelles like lysosomes and mitochondria, which co-traffic with RNA-binding proteins (RBPs). While hundreds of RNAs are known to be enriched in these structures, the molecular mechanisms governing their localization remain unclear. In polarized cells like neurons, mRNA localization and translation in specific subcellular regions and organelles help establish and maintain specialized domains necessary for neural circuit communication through dendrites and axons (Mandal & Drerup, 2019).

In this work, Lo and colleagues address the knowledge gap regarding how RNA localization influences cellular function by investigating the role of the RNA-binding protein HNRNPA2B1 in regulating RNA localization and its impact on cellular function, specifically in neurons.

Key Findings

HNRPA2B1 directs motor protein coding RNAs subcellular localization in neurons: Firstly, the authors explored the differential expression of RNAs after the loss and rescue of HNRNPA2B1. In HNRNPA2B1 KO conditions, an increased motor protein-related mRNA abundance in neurites was evident, suggesting its basal role in excluding RNA from these neuronal projections. The authors showed the effects on both kinesin and dynein on RNA abundance (Figs. 1 – 2) with RNA seq analysis and single molecule FISH microscopy.

The authors found that 3’UTRs of RNAs whose localization was sensitive to HNRNPA2B1 were enriched for AG-rich sixmers, which are known to bind HNRNPA2B1 (Fig.3). This suggests that the protein may directly bind these RNAs to regulate their localization.They examined HNRNPA2B1 CLIP data and found that RNAs whose localization was sensitive to the protein were more likely to be bound by it. Finally, related to RNA localization by HNRPA2B1 function, the authors show that HNRNPA2B1 point mutations that increase cytoplasmic abundance led to stronger suppression of RNA mislocalization defects, in part, proving that HNRPA2B1 localization is tightly related with their target RNAs spatial restriction (Fig. 4)

Motor protein function is hindered upon loss of HNRPA2B1 expression: Using live cell microscopy, the authors noted that HNRNPA2B1 dysfunction affects motor protein activity, as observed through lysosome movement in neurites (Fig. 5). In KO conditions, lysosomes appeared less motile in either direction of microtubule ends. Importantly, there are no changes in the soma of neurons for these experiments.

A proposed mechanism for HNRPA2B1 regulating RNA stability: CAF1 is an enzymatic subunit of the CCR4-NOT deadenylase, which is recruited by HNRNPA2B1 to promote RNA degradation. CAF1 inactivation was achieved through the expression of a dominant-negative version of the protein. Results show that HNRNPA2B1-target RNAs were preferentially stabilized after the loss of CCR4-NOT deadenylase activity, supporting the idea that HNRNPA2B1 negatively regulates RNA stability via CCR4-NOT recruitment. HNRNPA2B1-target RNAs became more enriched in neurites following the loss of CCR4-NOT activity, compared to RNAs insensitive to HNRNPA2B1. The authors propose that HNRNPA2B1 binding is linked to CAF1-mediated RNA stability regulation.

What I like

Use of inducible rescue and mutation models: The use of knockout models, subcellular fractionation analysis, and live-cell microscopy is useful and sound for the analysis performed. The mRNA candidates (Kinesin and dynein components) are also relevant to show the consequences of HNRNPA2B1 presence on motor protein-related transcripts. I liked how the data is presented, and that the way in which the analyses were performed remains clear throughout the manuscript.

Generalized mechanism: This study suggests that RNA stability regulation by RBPs may be a common mechanism in subcellular RNA localization. The findings shed light on a subject that could be further explored, and it will be interesting to understand how local translation is regulated not only in neuronal models but other cells.

Neurodegenerative diseases: HNRNPA2B1 dysfunction may be linked to disorders such as Alzheimer’s or ALS, given its impact on RNA stability and transport and the subsequential effect in local translation.

Future directions and questions

It is important to note that the authors state in several parts of this preprint that they are suggesting functional links between HNRPNA2B1 and its RNA targets while not showing direct evidence of cellular and molecular mechanisms. Additionally, one could propose future directions and question the following:

Future Directions

Isoform-Specific Functions of HNRNPA2B1: Future studies should explore the distinct roles of HNRNPA2B1 isoforms using true wild-type cells, as this could reveal unique contributions to RNA localization and stability.
Explore Disease-Associated Mutations: Investigating HNRNPA2B1 mutations linked to neurodegenerative diseases could reveal if and how these mutations disrupt RNA localization or motor protein function. Such research may provide critical insights into the role of HNRNPA2B1 in diseases like Alzheimer’s and ALS.
RNA-Motor Protein Co-Trafficking: Investigating the co-trafficking of RNA and motor proteins through live-cell imaging could help confirm whether HNRNPA2B1 directly influences motor protein function by regulating RNA localization.

Questions

What are the Isoform-Specific Functions? How do different HNRNPA2B1 isoforms affect RNA localization and stability, and do they perform complementary or distinct functions?

Is There a Direct Link Between RNA and Motor Protein Activity? Does the increase in motor protein-related RNA directly cause the observed reduction in motor protein activity, or is there an intermediary process?

How Does HNRNPA2B1 Regulate RNA Stability? What precise mechanisms does HNRNPA2B1 employ to regulate RNA stability, particularly through its interaction with CAF1 and other components?

References

Das, S., Vera, M., Gandin, V., Singer, R. H., & Tutucci, E. (2021). Intracellular mRNA transport and localized translation. Nature Reviews Molecular Cell Biology Nat Rev Mol Cell Biol, 22(7), 483-504.

Lin, J. Q., van Tartwijk, F. W., & Holt, C. E. (2021). Axonal mRNA translation in neurological disorders. RNA Biology, 18(7), 936-961.

Mandal, A., & Drerup, C. M. (2019). Axonal Transport and Mitochondrial Function in Neurons. Frontiers in Cellular Neuroscience, 13.

Tags: dynein, hnrnpa2, hnrnpb1, kinesin, motor protein, neurons, rna, rna localization

doi: https://doi.org/10.1242/prelights.38476

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(1 votes)

Author's response

Dr. Matthew Taliaferro shared

1. Regarding question 1, we did look at the two different main isoforms (HNRNPA2 and HNRNPB1) separately, and didn’t see much difference in the RNAs that they regulated the localization of. So I think these two isoforms are probably interchangeable, at least as far as RNA localization is concerned. It’s totally possible they have different functions for other RNA-based processes (splicing, etc.).

2. For question 2, I totally agree! Whether there is a direct link between RNA and motor protein activity is not known, and if there was one, how would it work? We are thinking about experiments to test this now, but it’s a rather tricky experiment to do as the methods for ectopically forcing RNAs to a particular location are difficult.

3. For question 3, there’s decent evidence that HNRNPA2B1 recruits CCR4-NOT (of which Caf1 is a member) (Geissler et al, Genes and Development, 2016), but I don’t think exactly how that works is known. This is also something we are gearing up to go after!

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