Most of the eukaryotic genome is transcribed, yielding a complex network of transcripts that includes tens of thousands of long noncoding RNAs with little or no protein-coding capacity. Although the vast majority of long noncoding RNAs have yet to be characterized thoroughly, many of these transcripts are unlikely to represent transcriptional “noise” as a significant number have been shown to exhibit cell type-specific expression, localization to subcellular compartments, and association with human diseases. Here, we highlight recent efforts that have identified a myriad of molecular functions for long noncoding RNAs. In some cases, it appears that simply the act of noncoding RNA transcription is sufficient to positively or negatively affect the expression of nearby genes. However, in many cases, the long noncoding RNAs themselves serve key regulatory roles that were assumed previously to be reserved for proteins, such as regulating the activity or localization of proteins and serving as organizational frameworks of subcellular structures. In addition, many long noncoding RNAs are processed to yield small RNAs or, conversely, modulate how other RNAs are processed. It is thus becoming increasingly clear that long noncoding RNAs can function via numerous paradigms and are key regulatory molecules in the cell.

Recent deep sequencing studies have revealed thousands of circular noncoding RNAs generated from proteincoding genes. These RNAs are produced when the precursor messenger RNA (pre-mRNA) splicing machinery ''backsplices'' and covalently joins, for example, the two ends of a single exon. However, the mechanism by which the spliceosome selects only certain exons to circularize is largely unknown. Using extensive mutagenesis of expression plasmids, we show that miniature introns containing the splice sites along with short (~30-to 40-nucleotide) inverted repeats, such as Alu elements, are sufficient to allow the intervening exons to circularize in cells. The intronic repeats must base-pair to one another, thereby bringing the splice sites into close proximity to each other. More than simple thermodynamics is clearly at play, however, as not all repeats support circularization, and increasing the stability of the hairpin between the repeats can sometimes inhibit circular RNA biogenesis. The intronic repeats and exonic sequences must collaborate with one another, and a functional 39 end processing signal is required, suggesting that circularization may occur post-transcriptionally. These results suggest detailed and generalizable models that explain how the splicing machinery determines whether to produce a circular noncoding RNA or a linear mRNA.

Studies of the transcriptional output of the human and mouse genomes have revealed that there are many more transcripts produced than can be accounted for by predicted protein-coding genes. Using a custom microarray, we have identified 184 non-coding RNAs that exhibit more than twofold up-or down-regulation upon differentiation of C2C12 myoblasts into myotubes. Here, we focus on the Men e/b locus, which is up-regulated 3.3-fold during differentiation. Two non-coding RNA isoforms are produced from a single RNA polymerase II promoter, differing in the location of their 39 ends. Men e is a 3.2-kb polyadenylated RNA, whereas Men b is an ;20-kb transcript containing a genomically encoded poly(A)-rich tract at its 39-end. The 39-end of Men b is generated by RNase P cleavage. The Men e/b transcripts are localized to nuclear paraspeckles and directly interact with NONO. Knockdown of MEN e/b expression results in the disruption of nuclear paraspeckles. Furthermore, the formation of paraspeckles, after release from transcriptional inhibition by DRB treatment, was suppressed in MEN e/b-depleted cells. Our findings indicate that the MEN e/b non-coding RNAs are essential structural/organizational components of paraspeckles.

MALAT1 is a long non-coding RNA whose expression level was originally identified as a predictor of metastasis of non-small cell lung tumors and was subsequently shown to be over-expressed in many human cancers. Here, we have identified a highly conserved tRNA-like small RNA of 61 nucleotides that originates from the MALAT1 locus and is broadly expressed in human tissues. In contrast to the long MALAT1 transcript that localizes to nuclear speckles, the small RNA exclusively localizes to the cytoplasm. We show that RNase P cleaves downstream of a genomically encoded poly(A)-rich tract to simultaneously generate the 3’ end of the abundant MALAT1 transcript and the 5’ end of the small RNA. Our findings reveal a novel 3’ end processing mechanism by which a single locus can yield both a stable nuclear retained non-coding RNA with a short poly(A) tail-like moiety and a small tRNA-like cytoplasmic RNA.

Thousands of eukaryotic protein-coding genes are noncanonically spliced to produce circular RNAs. Bioinformatics has indicated that long introns generally flank exons that circularize in Drosophila, but the underlying mechanisms by which these circular RNAs are generated are largely unknown. Here, using extensive mutagenesis of expression plasmids and RNAi screening, we reveal that circularization of the Drosophila laccase2 gene is regulated by both intronic repeats and trans-acting splicing factors. Analogous to what has been observed in humans and mice, basepairing between highly complementary transposable elements facilitates backsplicing. Long flanking repeats (∼400 nucleotides [nt]) promote circularization cotranscriptionally, whereas pre-mRNAs containing minimal repeats (<40 nt) generate circular RNAs predominately after 3 ′ end processing. Unlike the previously characterized Muscleblind (Mbl) circular RNA, which requires the Mbl protein for its biogenesis, we found that Laccase2 circular RNA levels are not controlled by Mbl or the Laccase2 gene product but rather by multiple hnRNP (heterogeneous nuclear ribonucleoprotein) and SR (serine-arginine) proteins acting in a combinatorial manner. hnRNP and SR proteins also regulate the expression of other Drosophila circular RNAs, including Plexin A (PlexA), suggesting a common strategy for regulating backsplicing. Furthermore, the laccase2 flanking introns support efficient circularization of diverse exons in Drosophila and human cells, providing a new tool for exploring the functional consequences of circular RNA expression across eukaryotes.