IncRNAs

What is a Long Non-Coding RNA?

Long non-coding (lnc) RNAs are defined as non-protein coding RNAs distinct from housekeeping RNAs such as tRNAs, rRNAs, and snRNAs, and independent from small RNAs with specific molecular processing machinery such as micro- or piwi-RNAs1.

The co-occurrence of massively parallel sequencing technology applied to RNA and the recognition that non-coding, functional RNA species may not be restricted to X-chromosome inactivation2 or to protein synthesis machinery, have revealed an RNA universe of remarkable diversity in plant and animal cells. Non-coding (nc) RNAs, those RNA molecules that are not templates for protein synthesis, make up a large portion of the total RNA in the cell suggesting a profound functional importance3. Genomes are extensively transcribed and give rise to thousands of long non-coding RNAs (lncRNAs), which are defined as RNAs longer than 200 nucleotides that are not translated into functional proteins. This broad definition encompasses a large and highly heterogeneous collection of transcripts that differ in their biogenesis and genomic origin4,5.

It is well documented that a growing number of lncRNAs have important cellular functions. The expression of a considerable number of lncRNAs is regulated and some have roles in different mechanisms of gene regulation. Several lncRNAs control the expression of nearby genes by affecting their transcription, and also affect other facets of chromatin biology, such as DNA replication or the response to DNA damage and repair. Other lncRNAs function away from their loci; their functions can be of a structural and/or regulatory nature and involve different stages of mRNA life, including splicing, turnover and translation, as well as signaling pathways. Consequently, lncRNAs affect several cellular functions that are of great physiological relevance, and alteration of their expression is inherent to numerous diseases. The specific expression patterns of these functional lncRNAs have the potential of being used as optimal disease biomarkers, and strategies are under development for their therapeutic targeting6.

Figure 1: The multiple dimensions of long noncoding RNA (lncRNA) function. lncRNAs can regulate gene expression by different mechanisms, some of which are illustrated here. These modes of action include lncRNA transcription-dependent activation or repression of neighbor genes (1), lncRNA-mediated inter-chromosomal interactions (2), formation of nuclear structures (i.e. paraspeckles) (3) or R-loops (4), lncRNAs as guide (5) or decoy (6) of transcription factors or as a scaffold for chromatin modifying complexes (7), lncRNAs acting as sponges of miRNAs (8), regulating post-transcriptional mRNA decay (9), regulating the cellular localization of RNA-binding proteins (RBPs) (10) or DNA-binding proteins (DBPs) (11)7.

Biogenesis and Cellular Fates of Long Non-Coding RNAs

Most lncRNA species are transcribed by Pol II. As such, many have 5′-end m7G caps and 3′-end poly(A) tails and are presumed to be transcribed and processed similarly to mRNAs. Importantly, distinct transcription, processing, export, and turnover of lncRNAs, which are closely linked with their cellular fates and functions.

Compared with mRNAs, a greater proportion of lncRNAs are localized in the nucleus8–10. Dissection of the global features of lncRNAs and mRNAs suggests that lncRNA genes are less evolutionarily conserved, contain fewer exons, and are less abundantly expressed. Early studies indicated that lncRNA genes likely contain fewer exons than mRNAs10–12. The recently developed RNA capture long seq enabled better annotation of the full length of lncRNAs, including their 5′ ends13,14, revealing little length difference with mRNAs, although lncRNAs contain fewer and longer exons. Single-cell sequencing found that some lncRNAs can be abundantly expressed in the human neocortex15.

Figure 2: a | Biogenesis of long non-coding RNAs (lncRNAs). Unlike mRNAs, many RNA polymerase II (Pol II)-transcribed lncRNAs are inefficiently processed and are retained in the nucleus (mechanisms of lncRNA nuclear retention are shown in parts be), whereas others are spliced and exported to the cytoplasm. The lncRNAs (and mRNAs) that contain one or only few exons are exported to the cytoplasm by nuclear RNA export factor 1 (NXF1). b | Some lncRNAs are transcribed by dysregulated Pol II, remain on chromatin and, subsequently, are degraded by the nuclear exosome. c | Numerous lncRNAs with a certain U1 small nuclear RNA (U1 snRNA) binding motif can recruit the U1 small nuclear ribonucleoprotein (U1 snRNP) and through it associate with Pol II at various loci. d | In many lncRNAs, the sequence between the 3′ splice site and the branch point is longer and contains a shorter polypyrimidine tract (PPT) than in mRNAs, which results in inefficient splicing. e | Sequence motifs in cis and factors in trans coordinately contribute to nuclear localization of lncRNAs. A nuclear retention element (NRE) U1 snRNA-binding site and C-rich motifs can recruit U1 snRNP and heterogeneous nuclear ribonucleoprotein K (hnRNPK), respectively, to enhance lncRNA nuclear localization. Other, differentially expressed RNA-binding proteins (RBPs), such as peptidylprolyl isomerase E (PPIE), inhibit splicing of groups of lncRNAs, resulting in their nuclear retention. f | In the cytoplasm, lncRNAs usually interact with diverse RBPs. g | Many lncRNAs in the cytoplasm are associated with ribosomes through ‘pseudo’ 5′ untranslated regions (UTRs); ribosome-associated lncRNAs tend to have short half-lives owing to unknown mechanisms. h | Several lncRNAs are sorted into mitochondria by unknown mechanisms. For example, the RNA component of mitochondrial RNA-processing endoribonuclease (RMRP) is recruited to mitochondria and is stabilized by binding G-rich RNA sequence-binding factor 1 (GRSF1). i | Some lncRNAs are also found in other organelles, such as exosomes, probably by forming lncRNA–RBP complexes. m7G, 7-methyl guanosine 5′ cap; (A)n, poly(A) 3′ tail6.

Like proteins, the function of lncRNAs depends on their subcellular localization/fates. Many lncRNAs are recognized as important modulators for nuclear functions2,16, and exhibit distinct nuclear localization patterns (Fig. 3a-d). Others must be exported to the cytoplasm to carry out their regulatory roles (Fig. 3e). In this review, several well-characterized lncRNAs are classified into three groups depending on their subcellular localization to illustrate the association of lncRNA localization and function: those that are absolutely nuclear localized in cis (Fig. 3a-b), those that are mainly nuclear localized and function in trans (Fig. 3c-d), and those that largely localize and function in the cytoplasm (Fig. 3e). It is worth noting that a recent large-scale evaluation of the subcellular fates of lncRNAs in human cell lines using single-molecule RNA fluorescence in situ hybridization revealed that lncRNAs exhibited a wide range of subcellular localization patterns, including not only distinct patterns of nuclear localization but also nonspecific location in both the nucleus and cytoplasm17.

Figure 3: Functions of Long Noncoding RNAs (lncRNAs) Are Associated with Their Subcellular Fates. lncRNAs have distinct subcellular localization patterns, allowing lncRNAs to execute their specified functions. (A) lncRNAs can accumulate and act in cis once they are transcribed. (B) lncRNAs can accumulate in cis once they are transcribed, but act in trans which affects genes located in the same chromosome at a distance or in different chromosomes. (A and B) Mechanisms of lncRNA in cis localization are largely unknown. (C) lncRNAs can localize elsewhere in the nucleoplasm in trans and act in trans. (D) lncRNAs can accumulate to specific nuclear bodies (orange circles) and act in trans. The mechanisms of lncRNA nuclear retention remain largely unknown, as does whether such lncRNAs are required to be translocated in the nucleus to regulate gene expression. (E) lncRNAs can be exported to the cytoplasm to execute their functions. For example, a cytoplasmic lncRNA can sequester protein (pink circle) or interfere with protein post-translational modification (PTM). Whether the nucleocytoplasmic export of cytoplasmic lncRNAs is distinct from that of mRNA has not yet been examined. The color of the shaded oval indicates differences in the protein composition of long noncoding ribonucleoproteins. Pink arrows, the unknown mechanisms for lncRNAs to gain specific subcellular localization patterns; black arrows, lncRNAs execute functions in distinct subcellular compartments. BCAR4, breast cancer antiestrogen resistance 4; CCAT1-L, colon cancer-associated transcript 1-long isoform; FIRRE, functional intergenic repeating RNA element; lincRNA, large intergenic noncoding RNAs; MALAT1, metastasis-associated lung adenocarcinoma transcript 1; NBs, nuclear bodies; NEAT1, nuclear-enriched abundant transcript 1; NKILA, nuclear factor-κB interacting lncRNA; NORAD, noncoding RNA activated by DNA damage; PVT1, plasmacytoma variant translocation 1; Sno-lncRNA, small nucleolar RNA-ended lncRNAs18.

Overall, lncRNAs are spliced less efficiently than mRNAs10,14,19. They have weaker internal splicing signals and longer distances between the 3′ splice site and the branch point14,20, which correlate with augmented nuclear retention10,14,19 (Fig. 2d). Other factors, such as differential expression of certain splicing regulators, also contribute to the accumulation of lncRNAs in the nucleus. For example, in mouse embryonic stem cells (mESCs), the highly expressed splicing inhibitor peptidylprolyl isomerase E suppresses the splicing of a subset of lncRNAs, leading to significant nuclear accumulation of many lncRNAs in mESCs10 (Fig. 2e). Alternative polyadenylation signals within lncRNAs may also modulate their subcellular localization. For example, the CCAT1 lncRNA gene produces two isoforms: the long isoform (CCAT1-L) is nuclear and contains an internal polyadenylation site corresponding with the 3′ ends of the short isoform (CCAT1-S), which is cytoplasmic21. Additionally, to these general features of lncRNA transcription and processing, lncRNAs often contain embedded sequence motifs that can recruit certain nuclear factors, which promote the nuclear localization and function of the lncRNA (Fig. 2e). For example, the lncRNA maternally expressed gene 3 (MEG3) contains a 356-nucleotide nuclear retention element that associates with U1 snRNP, which in turn retains MEG3 in the nucleus22.

Simply put, lncRNAs are a large and diverse class of transcripts that affect gene regulation through a variety of mechanisms. Depending on their genomic origin, subcellular localization, or functional pathways, lncRNAs can be classified into different groups. Like proteins, lncRNAs must localize to specific subcellular compartments to execute their functions. The nuclear localization and fate of lncRNAs are coordinately regulated at multiple layers, from transcription and processing to nuclear export through multiple sequence motifs in cis and factors in trans. However, how the specific localization of lncRNAs is achieved and regulated and what rules lncRNAs follow to make them so remarkably different from mRNAs remain largely unknown. In addition, the nucleocytoplasmic export of cytoplasmic lncRNAs and their life cycle in the cytoplasm also require a thorough investigation. Nevertheless, understanding these features of lncRNAs will greatly expand our current knowledge of lncRNA biology and shed new light into the study of their cellular roles in depth18.

References

  1. Ernst, C. & Morton, C. C. Identification and function of long non-coding RNA. Front. Cell. Neurosci. 7, (2013).
  2. Batista, P. J. & Chang, H. Y. Long Noncoding RNAs: Cellular Address Codes in Development and Disease. Cell 152, 1298–1307 (2013).
  3. Novikova, I. V., Hennelly, S. P., Tung, C.-S. & Sanbonmatsu, K. Y. Rise of the RNA Machines: Exploring the Structure of Long Non-Coding RNAs. Journal of Molecular Biology 425, 3731–3746 (2013).
  4. Uszczynska-Ratajczak, B., Lagarde, J., Frankish, A., Guigo, R. & Johnson, R. Towards a complete map of the human long non-coding RNA transcriptome. Nat. Rev. Genet. 19, 535–548 (2018).
  5. Fang, S. et al. NONCODEV5: a comprehensive annotation database for long non-coding RNAs. Nucleic Acids Research 46, D308–D314 (2018).
  6. Statello, L., Guo, C.-J., Chen, L.-L. & Huarte, M. Gene regulation by long non-coding RNAs and its biological functions. Nat Rev Mol Cell Biol 22, 96–118 (2021).
  7. Marchese, F. P., Raimondi, I. & Huarte, M. The multidimensional mechanisms of long noncoding RNA function. Genome Biol 18, 206 (2017).
  8. Derrien, T. et al. The GENCODE v7 catalog of human long noncoding RNAs: Analysis of their gene structure, evolution, and expression. Genome Res. 22, 1775–1789 (2012).
  9. Tian, B. & Manley, J. L. Alternative polyadenylation of mRNA precursors. Nat Rev Mol Cell Biol 18, 18–30 (2017).
  10. Guo, C.-J. et al. Distinct Processing of lncRNAs Contributes to Non-conserved Functions in Stem Cells. Cell 181, 621-636.e22 (2020).
  11. Quinn, J. J. et al. Rapid evolutionary turnover underlies conserved lncRNA–genome interactions. Genes Dev. 30, 191–207 (2016).
  12. Hezroni, H. et al. Principles of Long Noncoding RNA Evolution Derived from Direct Comparison of Transcriptomes in 17 Species. Cell Reports 11, 1110–1122 (2015).
  13. Lagarde, J. et al. High-throughput annotation of full-length long noncoding RNAs with capture long-read sequencing. Nat Genet 49, 1731–1740 (2017).
  14. Melé, M. et al. Chromatin environment, transcriptional regulation, and splicing distinguish lincRNAs and mRNAs. Genome Res. 27, 27–37 (2017).
  15. Liu, S. & Trapnell, C. Single-cell transcriptome sequencing: recent advances and remaining challenges. F1000Res 5, 182 (2016).
  16. Chen, L.-L. & Carmichael, G. G. Decoding the function of nuclear long non-coding RNAs. Current Opinion in Cell Biology 22, 357–364 (2010).
  17. Cabili, M. N. et al. Localization and abundance analysis of human lncRNAs at single-cell and single-molecule resolution. Genome Biol 16, 20 (2015).
  18. Chen, L.-L. Linking Long Noncoding RNA Localization and Function. Trends in Biochemical Sciences 41, 761–772 (2016).
  19. Zuckerman, B. & Ulitsky, I. Predictive models of subcellular localization of long RNAs. RNA 25, 557–572 (2019).
  20. Rosenberg, A. B., Patwardhan, R. P., Shendure, J. & Seelig, G. Learning the Sequence Determinants of Alternative Splicing from Millions of Random Sequences. Cell 163, 698–711 (2015).
  21. Xiang, J.-F. et al. Human colorectal cancer-specific CCAT1-L lncRNA regulates long-range chromatin interactions at the MYC locus. Cell Res 24, 513–531 (2014).
  22. Azam, S. et al. Nuclear retention element recruits U1 snRNP components to restrain spliced lncRNAs in the nucleus. RNA Biology 16, 1001–1009 (2019).

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