Alternative Splicing of Neuronal Differentiation Factor TRF2 Regulated by HNRNPH1/H2

Abstract

SUMMARY During neuronal differentiation, use of an alternative splice site on the rat telomere repeat-binding factor 2 (TRF2) mRNA generates a short TRF2 protein isoform (TRF2-S) capable of derepressing neuronal genes. However, the RNA-binding proteins (RBPs) controlling this splicing event are unknown. Here, using affinity pulldown analysis, we identified HNRNPH (heterogeneous nuclear ribonucleoprotein H) proteins as RBPs specifically capable of interacting with the spliced RNA segment (exon 7) of Trf2 pre-mRNA. HNRNPH proteins prevent the production of the short isoform of Trf2 mRNA, as HNRNPH silencing selectively elevates TRF2-S levels. Accordingly, HNRNPH levels decline while TRF2-S levels increase during neuronal differentiation. In addition CRISPR/Cas9-mediated deletion of hnRNPH2 selectively accelerates the NGF-triggered differentiation of rat pheochromocytoma cells into neurons. In sum, HNRNPH is a splicing regulator of Trf2 pre-mRNA that prevents the expression of TRF2-S, a factor implicated in neuronal differentiation.

The long and the short of TRF2 in neurogenesis

“…While studying this question, we discovered a C-terminal truncated TRF2 splicing isoform (TRF2-S) that was expressed as neurons differentiated and grew their axons and dendrites, 17 and 5 years later we identified the splicing factor HNRNPH as being important for regulating neuronal TRF2 alternative splicing. 18 We also reported recently that instead of telomeric DNA, TRF2-S displayed a preference for binding RNA, and that TRF2-S is present in axons where it regulated mRNA trafficking. 19 In the present Extra Views, we briefly review the distinct functions of the nuclear, telomere-protective TRF2, and the cytoplasmic RNA-regulatory TRF2-S. We discuss the generation of the two TRF2 splicing isoforms and the role of HNRNPH in regulating the production of TRF2 in neural stem cells (NSCs) and TRF2-S in mature neurons.…”

“…Among the RBPs that were pulled down from rodent brain lysate, HNRNPH1 and HNRNPH2 (2 highly conserved proteins, >95%) positively regulated exon 7 inclusion and inhibited TRF2-S production, although HNRNPH1 appeared to suppress splicing and TRF2-S production more robustly. 18 Crosslinking and immunoprecipitation revealed that human HNRNPH1 also bound TRF2 pre-mRNA, and that the binding sequence was a G-rich tract containing A residues. 53,54 HNRNPH binding sites on and near exon 7 were further identified using in vitro binding assays.…”

“…Interestingly, 3 binding sites were identified as potentially regulating splicing and all 3 appeared to be partially redundant. 18 Both paralogs were implicated in regulating alternative splicing. The significance of HNRNPH in controlling alternative splicing of neural genes was previously shown in oligodendrocytes.…”

“…46,49 A dominant negative form of TRF2 triggers a DNA damage response and senescence in mitotic neural cells and TRF2 inhibition enhances the differentiation of hippocampal neurons. 50 Molecular switch of TRF2 to TRF2-S by alternative splicing Alternative splicing of the TRF2 mRNA (Trf2 mRNA in rodents) leads to the production of a short protein isoform named TRF2-S. 18 Alternative splicing is a regulated process because recognition and recruitment of the spliceosomal machinery is suboptimal on alternative splice sites. 51 Trf2 mRNA contains 10 exons.…”

hnRNPs Interacting with mRNA Localization Motifs Define AxoNAl RNA Regulons

“…Both the RIP-seq data and depletion studies point to functions in axonal growth for hnRNP H1, F, and K. It will be of high interest to determine the molecular mechanisms for growth promotion by these hnRNPs and distinguish nuclear and somatic vs. axonal functions for these proteins. A few hnRNPs have been shown to impact neuronal differentiation and axon development (55)(56)(57). However, no studies have considered subcellular functions for these hnRNPs.…”