Our genomes contain the blueprint of what makes us human and many indications as to why we develop disease. Until the last 10 years, most studies had focussed on protein-coding genes, more specifically DNA sequences coding for proteins. However, this represents less than 5% of our genomes. The other 95% is referred to as the 'dark matter' of our genomes, our understanding of which is extremely limited. Part of this 'dark matter' includes regions that give rise to RNAs that do not code for proteins. A subset of these non-coding RNAs are long non-coding RNAs (lncRNAs), which in particular are beginning to be dissected and their importance to human health revealed. To improve our understanding and treatment of disease it is vital that we understand the molecular and cellular function of lncRNAs, and how their misregulation can contribute to disease. It is not yet clear what proportion of lncRNAs is actually functional; conservation during evolution is being used to understand the biological importance of lncRNA. Here, we present key themes within the field of lncRNAs, emphasising the importance of their roles in both the nucleus and the cytoplasm of cells, as well as patterns in their modes of action. We discuss their potential functions in development and disease using examples where we have the greatest understanding. Finally, we emphasise why lncRNAs can serve as biomarkers and discuss their emerging potential for therapy.No conflicts of interest were declared. What are lncRNAs?LncRNAs are RNAs of >200 nucleotides (nt) in length that are not thought to code for proteins. Although our appreciation and understanding of lncRNA function and importance has exploded in the last decade, the first lncRNAs were discovered in the 1990s: BC200, H19 [1], and Xist [2]. In the post-genomic era, extensive and deep RNA-Seq has revealed the existence of huge numbers of novel RNA transcripts, including lncRNAs. Many of these novel transcripts are low in abundance and so were not previously identified. Several consortia have been responsible for sequencing RNA from a variety of tissues, cell types, organisms, and disease states, and we now have a much more precise view of which RNA transcripts are expressed, and when and where (GENCODE [3], GTEX [4], FANTOM [5]).

The expression of long non-coding RNAs is highly enriched in the human nervous system.However, the function of neuronal lncRNAs in the cytoplasm and their potential translation remains poorly understood. Here we performed Poly-Ribo-Seq to understand the interaction of lncRNAs with the translation machinery and the functional consequences during neuronal differentiation of human SH-SY5Y cells. We discovered 237 cytoplasmic lncRNAs upregulated during early neuronal differentiation, 58-70% of which are associated with polysome translation complexes. Amongst these polysome associated lncRNAs, we find 45 small ORFs to be actively translated, 17 specifically upon differentiation. 15/45 of the translated lncRNA-smORFs exhibit sequence conservation within Hominidea suggesting they are under strong selective constraint in this clade. Profiling of publicly available datasets revealed that 8/45 of the translated lncRNAs are dynamically expressed during human brain development and 22/45 are associated with cancers of the central nervous system. One translated lncRNA we discovered is LINC01116, which is induced upon differentiation and contains an 87 codon smORF exhibiting increased ribosome profiling signal upon differentiation. The resulting LINC01116 peptide localises to neurites. Knockdown of LINC01116 results in a significant reduction of neurite length in differentiated cells indicating it contributes to neuronal differentiation. Our findings indicate cytoplasmic lncRNAs interact with translation complexes, are a non-canonical source of novel

Our understanding of mRNA translation and its regulation has been transformed by the development of ribosome profiling. This approach relies upon RNase footprinting of translating ribosomes in a precise manner to generate an accurate snapshot of ribosome positions with nucleotide resolution. Here we tested a variety of conditions, which contribute to the preciseness of ribosome footprinting and therefore the success of ribosome profiling. We found that NaCl concentration, RNaseI source, RNaseI amount, and temperature of footprinting all contributed to the quality of ribosome footprinting in human neuroblastoma SH-SY5Y cells. These ideal conditions for footprinting also improved footprint quality when used with Drosophila melanogaster S2 cells. Footprinting under the same conditions generated different footprints sizes and framing patterns in human and D. melanogaster cells. We also found that treatment of S2 cells with cycloheximide prior to footprinting impacted the distribution of footprints across ORFs, without affecting overall read length distribution and framing pattern, as previously found in other organisms. Together our results indicate that a variety of factors affect ribosome footprint quality and the nature of precise footprinting varies across species.

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