ICLIP identifies novel roles for SAFB1 in regulating RNA processing and neuronal function.

Caroline Rivers,James B Uney,Jessica Gaunt,Michael Norman,Youn-Bok Lee,Mark Rogers,Tomaz Curk,Leonidas Phylactou,Helen Scott,Jalilah Idris,Colin Campbell,Jernej Ule

doi:10.1186/s12915-015-0220-7

Abstract

BackgroundSAFB1 is a RNA binding protein implicated in the regulation of multiple cellular processes such as the regulation of transcription, stress response, DNA repair and RNA processing. To gain further insight into SAFB1 function we used iCLIP and mapped its interaction with RNA on a genome wide level.ResultsiCLIP analysis found SAFB1 binding was enriched, specifically in exons, ncRNAs, 3’ and 5’ untranslated regions. SAFB1 was found to recognise a purine-rich GAAGA motif with the highest frequency and it is therefore likely to bind core AGA, GAA, or AAG motifs. Confirmatory RT-PCR experiments showed that the expression of coding and non-coding genes with SAFB1 cross-link sites was altered by SAFB1 knockdown. For example, we found that the isoform-specific expression of neural cell adhesion molecule (NCAM1) and ASTN2 was influenced by SAFB1 and that the processing of miR-19a from the miR-17-92 cluster was regulated by SAFB1. These data suggest SAFB1 may influence alternative splicing and, using an NCAM1 minigene, we showed that SAFB1 knockdown altered the expression of two of the three NCAM1 alternative spliced isoforms. However, when the AGA, GAA, and AAG motifs were mutated, SAFB1 knockdown no longer mediated a decrease in the NCAM1 9–10 alternative spliced form. To further investigate the association of SAFB1 with splicing we used exon array analysis and found SAFB1 knockdown mediated the statistically significant up- and downregulation of alternative exons. Further analysis using RNAmotifs to investigate the frequency of association between the motif pairs (AGA followed by AGA, GAA or AAG) and alternative spliced exons found there was a highly significant correlation with downregulated exons. Together, our data suggest SAFB1 will play an important physiological role in the central nervous system regulating synaptic function. We found that SAFB1 regulates dendritic spine density in hippocampal neurons and hence provide empirical evidence supporting this conclusion.ConclusionsiCLIP showed that SAFB1 has previously uncharacterised specific RNA binding properties that help coordinate the isoform-specific expression of coding and non-coding genes. These genes regulate splicing, axonal and synaptic function, and are associated with neuropsychiatric disease, suggesting that SAFB1 is an important regulator of key neuronal processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0220-7) contains supplementary material, which is available to authorized users.

Highlights

Scaffold Attachment Factor B1 (SAFB1) is a RNA binding protein implicated in the regulation of multiple cellular processes such as the regulation of transcription, stress response, DNA repair and RNA processing
SAFB1 iCLIP analyses The sequences of SAFB1 and SAFB2 share considerable identity and we assessed the specificity of the anti-SAFB1 antibody
In the absence of UV crosslinking, 32P-end-labelled RNA was not immunoprecipitated with SAFB1 (Additional file 1). iCLIP was performed with partial RNase I digestion and, following SDS-PAGE analysis of immunoprecipitated SAFB1, the cross-linked RNA was extracted, reverse-transcribed and PCR-amplified before being subjected to high-throughput sequencing using

Summary

Introduction

SAFB1 is a RNA binding protein implicated in the regulation of multiple cellular processes such as the regulation of transcription, stress response, DNA repair and RNA processing. The scaffold attachment factor protein family consists of SAFB1, SAFB2 and a RBP termed SAFlike transcription modulator (SLTM) [3] They are large multi-domain proteins encoding a SAF box (DNA binding domain), an RNA recognition motif and Glu/Arg regions that mediate protein-protein interactions [3, 4]. Scaffold Attachment Factor B1 (SAFB1) is an evolutionarily conserved nuclear protein that has been associated with multiple cellular processes such as transcription, chromatin structure and apoptosis [4, 5]. SAFB1 was first identified attached to the scaffold/matrix attachment regions of chromatin [8] It was subsequently identified as HET/SAFB1, a protein that downregulates the transcription of the hsp gene [9] and hnRNPA1 associated protein, a factor recruited to nuclear stress bodies [10]. It has been hypothesised that nuclear stress bodies may sequester splicing factors to downregulate normal cellular splicing and facilitate the processing of RNA transcripts that are essential for the stress response [10, 24]

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