3/6/2023 0 Comments Obscure 2 code billiards roomAlthough they have become an experimental pillar of studies in gene regulation, a dedicated high-throughput systematic attempt to dissect the regulatory logic of subcellular RNA localization is still lacking. Large-scale testing of rationally designed or random sequence libraries has immensely contributed to elucidating the regulatory grammar of transcription ( 26–30), splicing ( 31–35), polyadenylation ( 36, 37), miRNA-mediated regulation ( 38), other forms of translational control ( 39, 40), and RNA nuclear enrichment and export ( 41–43) demonstrating the power and universal applicability of such approaches. Apart from a very limited number of such cases with a transparent link between a specific motif and a trans-acting factor mediating RNA sorting, the link between sequence and localization potential remains obscure and we still lack a systematic understanding of how the transcript sorting machinery works in a sequence-specific manner. The localization of beta-actin mRNA to the leading edge in fibroblasts and to axonal growth cones in neurons are well-studied examples of RNA localization ( 24): they have been found to be mediated by the RBP ZBP1 binding to a ‘zip-code’ in the three prime untranslated region (3′UTR) of the beta-actin mRNA ( 25). Alternatively, localized RBPs have been shown to prevent RNA degradation ( 23) or to capture and anchor transcripts and thereby create subcellular asymmetry in RNA localization ( 19). Several well-characterized localized transcripts are linked by RBPs to molecular motors, which enable active transport on the cytoskeleton ( 22). Approximately 1500 RBPs, interacting with a variety of RNA molecules, have been identified in mammalian cells ( 21). Most studied RNA localization phenotypes depend on interactions between RNA binding proteins (RBP) and the 3′ untranslated region (3′UTR) of a gene ( 19, 20). The extent of this phenomenon in steady-state and its dynamic adaptation in physiology ( 18) suggest that RNA localization is a tightly regulated process. This body of work reveals a wealth of mRNA localization patterns. Recent advances in spatial transcriptomics have enabled the study of dendritically localized RNAs at single cell resolution ( 15), with massively parallel hybridization approaches ( 16), and with expansion sequencing ( 17). Fractionation-based approaches allowed for the characterization of the entire pool of dendritically localized mRNAs ( 10, 11), revealing the richness of the local transcriptome and subsequently proteome ( 12, 13) as well as isoform-specific regulation ( 14). RNA localization in neurons was first demonstrated using in situ hybridization techniques ( 7, 8) and also revealed differences in the localization characteristics between neuronal cell types and brain regions ( 9). Neurons show a high degree of functional compartmentalization, which to a large part is achieved through transporting specific transcripts into dendrites or axons, where they are available for local (and sometimes activity-dependent) translation ( 6). mRNA localization might be an energy-efficient way to generate corresponding protein gradients, it might prevent harmful protein effects by ectopic activity or accumulation, and it could accelerate cellular response to extrinsic stimuli by activation of localized protein translation ( 5). Asymmetric subcellular mRNA distributions have been observed in a variety of polar cell types ( 1–4). The cytoplasm is a tightly regulated space that accommodates countless parallel tasks in specialized compartments. Testing this predictor on native mRNA sequencing data showed good agreement between predicted and observed localization potential, suggesting that the rules uncovered by our MPRA also apply to the localization of native full-length transcripts. Based on our data, we devised machine learning models that were able to predict the localization behavior of novel reporter sequences. We characterized the interplay between RNA stability and localization and identified motifs able to bias localization towards neurite or soma as well as the trans-acting factors required for their action. Mapping the localization potential of >300 genes revealed two ways neurite targeting can be achieved: focused localization motifs and broadly encoded localization potential. Here, we combined subcellular transcriptomics and massively parallel reporter assays and tested ∼50 000 sequences for their ability to localize to neurites. However, it is largely unknown how transcript sorting works in a sequence-specific manner. In neurons, localization of specific mRNAs to neurites is essential for cellular functioning. Asymmetric subcellular mRNA localization allows spatial regulation of gene expression and functional compartmentalization.
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