CLIP-Seq and massively parallel functional analysis of the CELF6 RNA binding protein reveals a role in destabilizing synaptic gene mRNAs through interaction with 3’UTR elements in vivo

Abstract

CELF6 is an RNA-binding protein in a family of proteins with roles in human health and disease, however little is known about the mRNA targets or in vivo function of this protein.We utilized HITS-CLIP/CLIP-Seq to identify, for the first time, in vivo targets of CELF6 and identify hundreds of transcripts bound by CELF6 in the brain. We found these are disproportionately mRNAs coding for synaptic proteins. We then conducted extensive functional validation of these targets, testing greater than 400 CELF6 bound sequence elements for their activity, applying a massively parallel reporter assay framework to evaluation of the CLIP data. We also mutated every potential CELF6 binding motif within these elements and tested their impact. This comprehensive analysis led us to ascribe a previously unknown function to CELF6: we found bound elements were generally repressive of translation, that CELF6 further enhances this repression via decreasing RNA abundance, and this process was dependent on UGU-rich sequence motifs. This greatly extends the known role for CELF6, which had previously been defined only as a splicing factor. We further extend these findings by demonstrating the same function for CELF3, CELF4, and CELF5. Finally, we demonstrate that the CELF6 targets are derepressed in CELF6 mutant mice in vivo, confirming this new role in the brain. Thus, our study demonstrates that CELF6 and other sub-family members are repressive CNS RNA-binding proteins, and CELF6 downregulates specific mRNAs in vivo.

Activity dependent translation in astrocytes dynamically alters the proteome of the perisynaptic astrocyte process

“…On the other hand, there could be a more non-specific mechanism where a variety of RBPs have redundant roles and work in a larger ensemble. For example, the set of RBPs identified as enriched in the upregulated transcripts is remarkably similar to the motifs found in our recent study of Celf6 binding targets (Rieger et al, 2018), even though the actual transcripts are distinct. As Celf6 and related Celf proteins have a role in forming RNA granules for mRNA storage and/or decay, and these granules often contain numerous species of RBPs and mRNAs interacting non-specifically through phase separation mediated by disordered domains, this suggests that transcripts enriched in binding for this selection of proteins may be more likely sequestered into a common granule awaiting some kind of signal for release and translation following neuronal activation.…”

Loss of CELF6 RNA binding protein impairs cocaine conditioned place preference and contextual fear conditioning

“…Much has been performed in this regard with FMRP, both through CLIP 51 and loss of function studies. 53 In contrast, the molecular targets of Celf6 are just now being studied, 54 and its molecular function is beginning to be elucidated. 5 Agents that decrease mGluR5 activity and its downstream signaling have rescued FXS-related phenotypes in Fmr1-deficient mouse models.…”

“…Many of the receptor signaling pathways that influence FMRP activity at the synapse, which in turn are dysregulated in the absence of FMRP, have been identified and pharmacologically targeted, such as NMDA, AMPA, BNDF and mTOR. 53 In contrast, the molecular targets of Celf6 are just now being studied, 54 and its molecular function is beginning to be elucidated. In in vitro assays it has been shown to have some activity as a splicing factor, 55 but localization in the central nervous system includes predominant cytoplasmic labeling, 4 consistent with non-nuclear roles in regulating RNA stability.…”

The Oft-Overlooked Massively Parallel Reporter Assay: Where, When, and Which Psychiatric Genetic Variants are Functional?

“…MPRAs quantify activity of putative regulatory elements by coupling them to a reporter gene and counting transcribed, element-specific tags ("barcodes"), using sequencing to determine the ratio of (expressed RNA barcode)/(delivered DNA barcode). This experimental framework has been applied to human splicing (21)(22)(23), RNA editing (24), protein translation (25), UTRs (26)(27)(28)(29)(30), and, most broadly, transcriptional (i.e., cis-) regulators. MPRAs thus offer a flexible framework to study regulatory phenomenon, including transcription factor (TF) and RNA binding protein (RBP) actions, transcript stability, and ribosome occupancy.…”

“…As shown in Figure 1C and 1D, the same architecture and RNA/DNA expression metric can be used to assess UTR effects on transcript stability. UTR MPRAs have yet to be implemented to study regulatory variants directly, but have been used to study the regulatory grammar of the ASD/ID-implicated CELF proteins and related RBPs (26,50,51), features conferring transcript stability via 3'UTRs (27,28,30), and 5'UTR influences on translation (52) and prediction of functional 5'UTR variants (29). Across enhancer and UTR MPRAs, several key forms of disease-associated noncoding variation can be assessed for functional consequences using a variety of model systems and delivery approaches (Figure 1E-F).…”