Weak binding to the A2RE RNA rigidifies hnRNPA2 RRMs and reduces liquid-liquid phase separation and aggregation.

Veronica H Ryan,Vincenzo Venditti,Mandar T Naik,Nicolas L Fawzi,Balabhadra Khatiwada,Joshua Amaya,Scott Watters

doi:10.1093/nar/gkaa710

Veronica H Ryan, Vincenzo Venditti + Show 5 more

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https://doi.org/10.1093/nar/gkaa710

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Abstract

hnRNPA2 is a major component of mRNA transport granules in oligodendrocytes and neurons. However, the structural details of how hnRNPA2 binds the A2 recognition element (A2RE) and if this sequence stimulates granule formation by enhancing phase separation of hnRNPA2 has not yet been studied. Using solution NMR and biophysical studies, we find that each of the two individual RRMs retain the domain structure observed in complex with RNA but are not rigidly confined (i.e. they move independently) in solution in the absence of RNA. hnRNPA2 RRMs bind the minimal rA2RE11 weakly but at least, and most likely, two hnRNPA2 molecules are able to simultaneously bind the longer 21mer myelin basic protein A2RE. Upon binding of the RNA, NMR chemical shift deviations are observed in both RRMs, suggesting both play a role in binding the A2RE11. Interestingly, addition of short A2RE RNAs or longer RNAs containing this sequence completely prevents in vitro phase separation of full-length hnRNPA2 and aggregation of the disease-associated mutants. These findings suggest that RRM interactions with specific recognition sequences alone do not account for nucleating granule formation, consistent with models where multivalent protein:RNA and protein:protein contacts form across many sites in granule proteins and long RNA transcripts.

Highlights

Spatial and temporal control of mRNA translation is crucial for highly coordinated biological processes like development and synaptic plasticity
The RNA-binding protein heterogeneous nuclear ribonucleoprotein A2 is a well-studied component of granules that move mRNA from the nucleus to sites of local translation [2]. hnRNPA2-containing granules were first studied in oligodendrocytes, where hnRNPA2 binds myelin basic protein mRNA and transports it to the oligodendroglial processes for local translation [3]. hnRNPA2 binds a cis-acting element in myelin basic protein mRNA called the A2 response element (A2RE) in the 3 untranslated region [3,4,5]
But not identical A2REs that bind hnRNPA2 have been identified in other transcripts, including mRNAs encoding ARC, neurogranin, alpha calcium calmodulin-dependent protein kinase II (␣CaMKII), tau, and brain derived neurotrophic factor (BDNF) [7,8,9], demonstrating that hnRNPA2 has the ability to bind a variety of sequences

Summary

Introduction

Spatial and temporal control of mRNA translation is crucial for highly coordinated biological processes like development and synaptic plasticity. But not identical A2REs that bind hnRNPA2 have been identified in other transcripts, including mRNAs encoding ARC, neurogranin, alpha calcium calmodulin-dependent protein kinase II (␣CaMKII), tau, and brain derived neurotrophic factor (BDNF) [7,8,9], demonstrating that hnRNPA2 has the ability to bind a variety of sequences. These transcripts are transported in transport granules containing mRNA, hnRNPA2 and other proteins [2]. Some evidence suggests that RNA nucleates stress granule formation, but it is not clear if this is true of transport granules [11,12,13]

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