Long noncoding RNAs: Re-writing dogmas of RNA processing and stability

Abstract

Most of the human genome is transcribed, yielding a complex network of transcripts that includes tens of thousands of long noncoding RNAs. Many of these transcripts have a 5' cap and a poly(A) tail, yet some of the most abundant long noncoding RNAs are processed in unexpected ways and lack these canonical structures. Here, I highlight the mechanisms by which several of these well-characterized noncoding RNAs are generated, stabilized, and function. The MALAT1 and MEN β (NEAT1_2) long noncoding RNAs each accumulate to high levels in the nucleus, where they play critical roles in cancer progression and the formation of nuclear paraspeckles, respectively. Nevertheless, MALAT1 and MEN β are not polyadenylated as the tRNA biogenesis machinery generates their mature 3' ends. In place of a poly(A) tail, these transcripts are stabilized by highly conserved triple helical structures. Sno-lncRNAs likewise lack poly(A) tails and instead have snoRNA structures at their 5' and 3' ends. Recent work has additionally identified a number of abundant circular RNAs generated by the pre-mRNA splicing machinery that are resistant to degradation by exonucleases. As these various transcripts use non-canonical strategies to ensure their stability, it is becoming increasingly clear that long noncoding RNAs may often be regulated by unique post-transcriptional control mechanisms. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa.

Keywords: CircRNA; Circular RNA; MALAT1; MEN β; NEAT1; Polyadenylation; Pre-mRNA splicing; RNA stability; Sno-lncRNA; Triple helix; tRNA-like.

Fig. 1

The MALAT1 locus generates a nuclear-retained long noncoding RNA and a tRNA-like small RNA. (A) Rather than using the canonical cleavage/polyadenylation machinery, the 3′ end of MALAT1 is almost always generated by tRNA biogenesis factors. First, RNase P cleavage simultaneously generates the mature 3′ end of MALAT1 and the 5′ end of mascRNA. The tRNA-like small RNA is subsequently cleaved by RNase Z, subjected to CCA addition, and exported to the cytoplasm. In contrast, the mature MALAT1 transcript localizes to nuclear speckles and its 3′ terminus is protected by a triple helical structure (PDB code 4PLX [84]). The mouse coordinates (GenBank accession number FJ209304) are given. (B) Mouse mascRNA adopts a tRNA-like cloverleaf secondary structure. CCA (denoted in red) is post-transcriptionally added by the CCA-adding enzyme. (C) As shown by the Multiz Alignment track of the UCSC Genome Browser, highly conserved A- and U-rich tracts are present immediately upstream of the MALAT1 RNase P cleavage site (denoted by arrow). (D) These conserved sequence motifs form base triplets (denoted by dashed lines) that protect the 3′ end of MALAT1 from degradation. A minimal triple helix that supports both RNA stability and translation [82] is shown. Nucleotides that function in promoting translation are denoted in purple. (E) U–A·U base triplets form via Hoogsteen hydrogen bonds to the major groove of a Watson–Crick base-paired helix.

Fig. 2

The MEN ε/β (NEAT1) locus is regulated by alternative 3′ end processing. (A) The MEN ε/β primary transcript can be cleaved by the canonical cleavage/polyadenylation machinery (to generate the polyadenylated MEN ε RNA) or by the tRNA biogenesis machinery (to generate the non-polyadenylated MEN β RNA). Analogous to the MALAT1 locus, an evolutionarily conserved triple helix is present immediately upstream of the RNase P cleavage site to protect the mature 3′ end of MEN β. The mouse coordinates (GenBank accession number GQ859163) are given. (B) The acceptor stem of the mouse MEN β tRNA-like small RNA is destabilized due to the presence of a C–A mismatch (denoted in red). This causes the CCA-adding enzyme to add CCACCA rather than CCA to the small RNA, triggering its efficient degradation.

Fig. 3

The pre-mRNA splicing machinery generates a number of non-polyadenylated noncoding RNAs. (A) When a snoRNA sequence is encoded in an intron, pre-mRNA splicing releases the excised intron, which is subsequently debranched and trimmed to produce the mature ~70–200 nt snoRNA. In this case, a box C/D snoRNA is shown. (B) In contrast, when two snoRNA sequences are present in a single intron, debranching and trimming of the excised intron produce a long noncoding RNA with snoRNA ends. (C) After splicing, some introns fail to be debranched and accumulate as stable circular intronic RNAs in the nucleus. These transcripts are covalent circles due to the 2′,5′-phosphodiester bond between the 5′ end of the intron and the branch point adenosine. (D) Pre-mRNA splicing can generate linear or circular RNAs comprised of exons. If the splice sites are joined in the canonical order, a mature linear mRNA is generated that is subsequently polyadenylated (top). Alternatively, the splicing machinery can backsplice and join a splice donor to an upstream splice acceptor, generating a circular RNA whose ends are covalently linked by a 3′,5′-phosphodiester bond (bottom).