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The identification of a new subclass of circular RNAs that are predominantly nuclear and promote transcription of their parental genes reveals a new regulatory function for these noncoding RNAs. Noncoding RNAs (ncRNAs) have numerous roles in development and disease, and one of the prominent roles is to regulate gene expression. A vast number of circular RNAs (circRNAs) have been identified, and some have been shown to function as microRNA sponges in animal cells. Here, we report a class of circRNAs associated with RNA polymerase II in human cells. In these circRNAs, exons are circularized with introns 'retained' between exons; we term them exon-intron circRNAs or EIciRNAs. EIciRNAs predominantly localize in the nucleus, interact with U1 snRNP and promote transcription of their parental genes. Our findings reveal a new role for circRNAs in regulating gene expression in the nucleus, in which EIciRNAs enhance the expression of their parental genes in cis , and highlight a regulatory strategy for transcriptional control via specific RNA-RNA interaction between U1 snRNA and EIciRNAs.

Intron retention has long been an exemplar of regulated splicing with case studies of individual events serving as models that provided key mechanistic insights into the process of splicing control. In organisms such as plants and budding yeast, intron retention is well understood as a major mechanism of gene expression regulation. In contrast, in mammalian systems, the extent and functional significance of intron retention have, until recently, remained greatly underappreciated. Technical challenges to the global detection and quantitation of transcripts with retained introns have often led to intron retention being overlooked or dismissed as “noise”. Now, however, with the wealth of information available from high-throughput deep sequencing, combined with focused computational and statistical analyses, we are able to distinguish clear intron retention patterns in various physiological and pathological contexts. Several recent studies have demonstrated intron retention as a central component of gene expression programs during normal development as well as in response to stress and disease. Furthermore, these studies revealed various ways in which intron retention regulates protein isoform production, RNA stability and translation efficiency, and rapid induction of expression via post-transcriptional splicing of retained introns. In this review, we highlight critical findings from these transcriptomic studies and discuss commonalties in the patterns prevalent in intron retention networks at the functional and regulatory levels.

Introns are removed by the spliceosome, a large macromolecular complex composed of five ribonucleoprotein subcomplexes (U snRNPs). The U1 snRNP, which binds to 5ʹ splice sites, plays an essential role in early steps of the splicing reaction. Here, we show that Arabidopsis thaliana LETHAL UNLESS CBC7 (LUC7) proteins, which are encoded by a three-member gene family in Arabidopsis, are important for plant development and stress resistance. We show that LUC7 is a U1 snRNP accessory protein by RNA immunoprecipitation experiments and LUC7 protein complex purifications. Transcriptome analyses revealed that LUC7 proteins are not only important for constitutive splicing, but also affect hundreds of alternative splicing events. Interestingly, LUC7 proteins specifically promote splicing of a subset of terminal introns. Splicing of LUC7-dependent introns is a prerequisite for nuclear export, and some splicing events are modulated by stress in a LUC7-dependent manner. Taken together, our results highlight the importance of the U1 snRNP component LUC7 in splicing regulation and suggest a previously unrecognized role of a U1 snRNP accessory factor in terminal intron splicing.

Chloroplasts play an essential role in plant growth and development through manipulating photosynthesis and the production of hormones and metabolites. Although many genes or regulators involved in chloroplast biogenesis and development have been isolated and characterized, identification of novel components is still lacking. We isolated a rice (Oryza sativa) mutant, termed albino leaf 2 (al2), using genetic screening. Phenotypic analysis revealed that the al2 mutation caused obvious albino leaves at the early developmental stage, eventually leading to al2 seedling death. Electron microscopy investigations indicated that the chloroplast structure was disrupted in the al2 mutants at an early developmental stage and subsequently resulted in the breakdown of the entire chloroplast. Molecular cloning illustrated that AL2 encodes a chloroplast group IIA intron splicing facilitator (CRS1) in rice, which was confirmed by a genetic complementation experiment. Moreover, our results demonstrated that AL2 was constitutively expressed in various tissues, including green and non-green tissues. Interestingly, we found that the expression levels of a subset of chloroplast genes that contain group IIA and IIB introns were significantly reduced in the al2 mutant compared to that in the wild type, suggesting that AL2 is a functional CRS1 in rice. Differing from the orthologous CRS1 in maize and Arabidopsis that only regulates splicing of the chloroplast group II intron, our results demonstrated that the AL2 gene is also likely to be involved in the splicing of the chloroplast group I intron. They also showed that disruption of AL2 results in the altered expression of chloroplast-associated genes, including chlorophyll biosynthetic genes, plastid-encoded polymerases and nuclear-encoded chloroplast genes. Taken together, these findings shed new light on the function of nuclear-encoded chloroplast group I and II intron splicing factors in rice.

Galactinol synthase (GolS) is the first committed enzyme in raffinose family oligosaccharide (RFO) synthesis pathway and synthesizes galactinol from UDP-galactose and inositol. Expression of GolS genes has long been implicated in abiotic stress, especially drought and salinity. A non-canonical regulation mechanism controlling the splicing and maturation of rice GolS genes was identified in rice photosynthetic tissue. We found that the two isoforms of Oryza sativa GolS (OsGolS) gene, located in chromosomes 3(OsGolS1) and 7(OsGolS2) are interspersed by conserved introns harboring characteristic premature termination codons (PTC). During abiotic stress, the premature and mature transcripts of both isoforms were found to accumulate in a rhythmic manner for very small time-windows interrupted by phases of complete absence. Reporter gene assay using GolS promoters under abiotic stress does not reflect this accumulation profile, suggesting that this regulation occurs post-transcriptionally. We suggest that this may be due to a surveillance mechanism triggering the degradation of the premature transcript preventing its accumulation in the cell. The suggested mechanism fits the paradigm of PTC-induced Nonsense-Mediated Decay (NMD). In support of our hypothesis, when we pharmacologically blocked NMD, the fulllength pre-mRNAs were increasingly accumulated in cell. To this end, our work suggests that a combined transcriptional and post transcriptional control exists in rice to regulate GolS expression under stress. Concurrent detection and processing of prematurely terminating transcripts coupled to repressed splicing can be described as a form of Regulated Unproductive Splicing and Translation (RUST) and may be linked to the stress adaptation of the plant, which is an interesting future research possibility.

The origin of the eukaryotic nucleus marked a seminal evolutionary transition. We propose that the nuclear envelope's incipient function was to allow mRNA splicing, which is slow, to go to completion so that translation, which is fast, would occur only on mRNA with intact reading frames. The rapid, fortuitous spread of introns following the origin of mitochondria is adduced as the selective pressure that forged nucleus–cytosol compartmentalization. Why have a nucleus? The discovery of introns, ‘junk’ sequences of DNA in the middle of genes that are excised before the mRNA is translated into a protein, prompted some serious thinking about the early evolution of the cell. William Martin and Eugene V. Koonin this week present a new idea to explain the first evolutionary step on the road to the eukaryotic nucleus. This assumes that the initial role of the nuclear envelope was as an intracellular filter to allow splicing, which is slow, to go to completion so that translation, which is fast, occurs only on fully processed mRNA.

Proper timing of gene expression is essential for many biological events, but the molecular mechanisms that control timing remain largely unclear. It has been suggested that introns contribute to the timing mechanisms of gene expression, but this hypothesis has not been tested with natural genes. One of the best systems for examining the significance of introns is the oscillator network in the somite segmentation clock, because mathematical modeling predicted that oscillating expression depends on negative feedback with a delayed timing. The basic helix–loop–helix repressor gene Hes7 is cyclically expressed in the presomitic mesoderm (PSM) and regulates the somite segmentation. Here, we found that introns lead to an ~19-min delay in the Hes7 gene expression, and mathematical modeling suggested that without such a delay, Hes7 oscillations would be abolished. To test this prediction, we generated mice carrying the Hes7 locus whose introns were removed. In these mice, Hes7 expression did not oscillate but occurred steadily, leading to severe segmentation defects. These results indicate that introns are indeed required for Hes7 oscillations and point to the significance of intronic delays in dynamic gene expression.

Bacterial group II introns encode maturase proteins required for splicing. In organelles of photosynthetic land plants, most of the group II introns have lost the reading frames for maturases. Here, we show that the plastidial maturase MatK not only interacts with its encoding intron within trnk-UUU, but also with six additional group II introns, all belonging to intron subclass llA. Mapping analyses of RNA binding sites revealed MatK to recognize multiple regions within the trnK intron. Organellar group ll introns are considered to be the ancestors of nuclear spliceosomal introns. That MatK associates with multiple intron ligands makes it an attractive model for an early trans-acting nuclear splicing activity.

The carotenoid cleavage dioxygenase 2, a new member of the CCD family, catalyzes the conversion of zeaxanthin into crocetin-dialdehyde in Crocus . CCD2 is expressed in flowers, being responsible for the yellow, orange and red colorations displayed by tepals and stigma. Three CsCCD2 genes were identified in Crocus sativus , the longest contains ten exons and the shorter is a truncated copy with no introns and which lacks one exon sequence. Analysis of RNA-seq datasets of three developmental stages of saffron stigma allowed the determination of alternative splicing in CsCCD2 , being intron retention (IR) the prevalent form of alternative splicing in CsCCD2 . Further, high IR was observed in tissues that do not accumulate crocetin. The analysis of one CsCCD2 promoter showed cis -regulatory motifs involved in the response to light, temperature, and circadian regulation. The light and circadian regulation are common elements shared with the previously characterized CsLycB2a promoter, and these shared common cis -acting elements may represent binding sites for transcription factors responsible for co-regulation of these genes during the development of the stigma in saffron. A daily coordinated rhythmic regulation for CsCCD2 and CsLycB2a was observed, with higher levels of mRNA occurring at low temperatures during darkness, confirming the results obtained in the in silico promoter analysis. In addition, to the light and temperature dependent regulation of CsCCD2 expression, the apocarotenoid β-cyclocitral up-regulated CsCCD2 expression and could acts as a mediator of chromoplast-to-nucleus signalling, coordinating the expression of CsCCD2 with the developmental state of the chromoplast in the developing stigma.

The highly conserved heat shock protein 70 (HSP70) contributes to survival at a cellular level and greatly enhances stress tolerance in many organisms. In this study, we isolate and characterize Cshsp702, which encodes an inducible form of HSP70 in the rice stem borer, Chilo suppressalis. Cshsp702 does not contain introns; the translational product is comprised of 629 amino acids with an isoelectric point of 5.69. Real-time quantitative PCR revealed that Cshsp702 was expressed at maximal levels in hemocytes and was minimally expressed in the midgut. Expression of Cshsp702 in response to a range of temperatures (-11 to 43 °C) indicated significant induction by extreme cold and hot temperatures, with maximum expression after 2 h at 42 °C. The induction of Cshsp702 in response to the endoparasite Cotesia chilonis was also studied; interestingly, Cshsp702 expression in C. suppressalis was significantly induced at 24 h and 5 days, which correspond to predicted times of C. chilonis feeding and growth, respectively. The potential induction of Cshsp702 as an inflammatory response due to parasitic stress is discussed. In conclusion, Cshsp702 is induced in response to both environmental and biotic stress and plays an important role in the physiological adaptation of C. suppressalis.