Abstract
The sorting of RNA molecules to subcellular locations facilitates the activity of spatially restricted processes. We have analyzed subcellular transcriptomes of FMRP-null mouse neuronal cells to identify transcripts that depend on FMRP for efficient transport to neurites. We found that these transcripts contain an enrichment of G-quadruplex sequences in their 3' UTRs, suggesting that FMRP recognizes them to promote RNA localization. We observed similar results in neurons derived from Fragile X Syndrome patients. We identified the RGG domain of FMRP as important for binding G-quadruplexes and the transport of G-quadruplex-containing transcripts. Finally, we found that the translation and localization targets of FMRP were distinct and that an FMRP mutant that is unable to bind ribosomes still promoted localization of G-quadruplex-containing messages. This suggests that these two regulatory modes of FMRP may be functionally separated. These results provide a framework for the elucidation of similar mechanisms governed by other RNA-binding proteins.
Highlights
All eukaryotic cells contain subcellular regions that are associated with specific functions
Identification of transcripts that depend upon FMRP for efficient neuronal transport We and others have previously used subcellular fractionation followed by high-throughput sequencing to quantify transcripts in the soma and neurites of neuronal cells (Taliaferro et al, 2016a; Zappulo et al, 2017)
We found genes that displayed reduced ribosome occupancy in FMRP null cells compared to wildtype cells were significantly more likely to have been identified as FMRP CLIP targets than expected (Figure 7B; Ascano et al, 2012; Darnell et al, 2011; Maurin et al, 2018), consistent with FMRP either generally acting to inhibit translation by inducing ribosome stalling or with FMRP generally acting as a translational activator (Greenblatt and Spradling, 2018)
Summary
All eukaryotic cells contain subcellular regions that are associated with specific functions. Precise molecular mechanisms that govern how transcripts are transported have been worked out in detail for only a small subset of heavily studied localized RNAs (Bertrand et al, 1998; Ephrussi et al, 1991; Jambhekar and Derisi, 2007; Kislauskis et al, 1994). Other studies have found that FMRP binds completely different short RNA motifs (Ray et al, 2013) while still others seem to show that FMRP displays almost no sequence specificity at all and instead binds all along the coding sequence of its target transcripts (Darnell et al, 2011) Whether or not these potentially different modes of binding are related to the different functions of FMRP is again unknown
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