Dissecting the role of cytoplasmic lncRNAs in neuronal differentiation and disease

Long non-coding RNAs (lncRNAs) account for ~30% of human genes and ~40% are specifically expressed in the brain. Their precise spatiotemporal expression patterns and dysregulation in neuronal disease suggests physiological and pathological relevance. Moreover, ribosome-profiling and proteomics have shown that many cytoplasmic lncRNAs contain unannotated or small ORFs (sORF) that can be translate into functional (micro)proteins. The Aspden group has previously performed Poly-Ribo-Seq on a human neuroblastoma cell line, SH-SY5Y, in immature and differentiated states identifying 45 translated ORFs within 35 annotated lncRNAs. These lncRNAs exhibited similar translational efficiencies to protein-coding genes.

To dissect the function of peptides translated from lncRNAs, a FLAG-tagged reporter assay was performed on 13 candidates selected based on their expression during neuronal development and dysregulation in disease. Of the translated lncRNAs investigated 6/13 produce proteins with subcellular localisations indicative of function, including nuclear and mitochondrial co-localisation. Additionally, siRNA knockdown of 4/6 translated lncRNAs resulted in dysregulation of neuronal differentiation, as characterised by the disruption of neuronal gene expression and attenuated neurite outgrowth. Of the 5 translated lncRNAs tested 4 were enriched in polysomes of cortical organoids, suggesting that they are translated in advanced models of human brain development.

One translated lncRNA, LIPT2-AS1, encodes a novel protein shown by FLAG-tagging assays in SH-SY5Y cells to localise to the nucleus. Knockdown of LIPT2-AS1-protein by CRISPR-Cas9 results in a significant reduction in SH-SY5Y neurite length. Furthermore, RNA-Seq identified a global downregulation of neuronal genes and an upregulation of genes associated with Proneural-Mesenchymal transition, demonstrating that LIPT2-AS1-protein is required for normal neuronal differentiation. Protein pulldown and Mass spectrometry revealed that LIPT2-AS1-protein interacts with nuclear proteins involved in transcription and RNA splicing.

Overall, this work identifies multiple translated lncRNAs required for neuronal differentiation and highlights the potential for lncRNA-derived proteins to expand the known proteome.