HnRNPs Interacting with mRNA Localization Motifs Define AxoNAl RNA Regulons

Seung Joon Lee,Juan A Oses-Prieto,Riki Kawaguchi,Pabitra K Sahoo,Amar N Kar,Meir Rozenbaum,David Oliver,Shreya Chand,Hao Ji,Michael Shtutman,Sharmina Miller-Randolph,Ross J Taylor,Mike Fainzilber,Giovanni Coppola,Alma L Burlingame,Jeffery L Twiss

doi:10.1074/mcp.ra118.000603

Abstract

mRNA translation in axons enables neurons to introduce new proteins at sites distant from their cell body. mRNA-protein interactions drive this post-transcriptional regulation, yet knowledge of RNA binding proteins (RBP) in axons is limited. Here we used proteomics to identify RBPs interacting with the axonal localizing motifs of Nrn1, Hmgb1, Actb, and Gap43 mRNAs, revealing many novel RBPs in axons. Interestingly, no RBP is shared between all four RNA motifs, suggesting graded and overlapping specificities of RBP-mRNA pairings. A systematic assessment of axonal mRNAs interacting with hnRNP H1, hnRNP F, and hnRNP K, proteins that bound with high specificity to Nrn1 and Hmgb1, revealed that axonal mRNAs segregate into axon growth-associated RNA regulons based on hnRNP interactions. Axotomy increases axonal transport of hnRNPs H1, F, and K, depletion of these hnRNPs decreases axon growth and reduces axonal mRNA levels and axonal protein synthesis. Thus, subcellular hnRNP-interacting RNA regulons support neuronal growth and regeneration.

Highlights

MRNA translation in axons enables neurons to introduce new proteins at sites distant from their cell body. mRNAprotein interactions drive this post-transcriptional regulation, yet knowledge of RNA binding proteins (RBP) in axons is limited
Axonal localization of Hmgb1 mRNA is driven through a 60 nt motif in its 3ЈUTR, and the Hmgb1 mRNA is constitutively transported into sensory axons [36]
Secondary structure predictions using aligned Nrn1 sequences show a stem-loop RNA structure for nt 95–188 compared with relatively unstructured nt 1–94. These findings indicate that Nrn1 mRNA’s nt 95–188 may serve as a binding motif for axonal RBPs

Summary

Introduction

MRNA translation in axons enables neurons to introduce new proteins at sites distant from their cell body. mRNAprotein interactions drive this post-transcriptional regulation, yet knowledge of RNA binding proteins (RBP) in axons is limited. MS analyses showed many proteins bound to localizing motifs of Nrn1 and Hmgb1 mRNAs. Replicate RAMS assays were normalized for protein yields across the individual pulldowns for target RNAs, control RNAs, and biotin controls (supplemental Table S2B–S2C).

Results

Conclusion