A single Drosophila Dscam1 genomic locus generates a vast repertoire of circRNAs facilitated by RNA pairing.

Mutually exclusive splicing is a regulated means to generate protein diversity, but the underlying mechanisms are poorly understood. Here comparative genome analysis revealed the built-in intronic elements for controlling mutually exclusive splicing of the 14-3-3ξ pre-mRNA. These elements are clade specific but are evolutionarily conserved at the secondary structure level. Combined evidence revealed the triple functions of these inter-intronic RNA pairings in synergistically ensuring the selection of only one of multiple exons, through activation of the proximal variable exon outside the loop by the approximation of cis elements, and simultaneous repression of the exon within the loop, in combination with the physical competition of RNA pairing. Additionally, under this model, we also deciphered a similar structural code in exon clusters 4 and 9 of Dscam (38,016 isoforms) and Mhc (480 isoforms). Our findings suggest a broadly applicable mechanism to ensure mutually exclusive splicing.

Alternative splicing of pre-mRNAs greatly contributes to diversity in products generated from a single gene. However, the underlying regulatory mechanisms are poorly understood. In the present study, we describe evolutionarily conserved intra-intronic and inter-intronic RNA secondary structures. Mutation experiments revealed that intra-intronic RNA secondary structure causes steric hindrance to enforce mutual splicing exclusivity, while inter-intronic RNA pairing largely functions through a looped-out mechanism. Moreover, mutually exclusive splicing may be regulated by RNA pairing competition between intra- and inter-introns. Importantly, the resulting dynamic RNA architecture largely controls mutually exclusive splicing, although cis-acting regulatory sequences may fine-tune this process. Our results emphasize the importance of dynamic RNA architecture in alternative splicing.

Mutually exclusive splicing is an important mechanism for expanding protein diversity. An extreme example is the Down syndrome cell adhesion molecular (Dscam1) gene of insects, containing four clusters of variable exons (exons 4, 6, 9, and 17), which potentially generates tens of thousands of protein isoforms through mutually exclusive splicing, of which regulatory mechanisms are still elusive. Here, we systematically analyzed the variable exon 4, 6, and 9 clusters of Dscam1 in Coleoptera species. Through comparative genomics and RNA secondary structure prediction, we found apparent evidence that the evolutionarily conserved RNA base pairing mediates mutually exclusive splicing in the Dscam1 exon 4 cluster. In contrast to the fly exon 6, most exon 6 selector sequences in Coleoptera species are partially located in the variable exon region. Besides, bidirectional RNA–RNA interactions are predicted to regulate the mutually exclusive splicing of variable exon 9 of Dscam1. Although the docking sites in exon 4 and 9 clusters are clade specific, the docking sites-selector base pairing is conserved in secondary structure level. In short, our result provided a mechanistic framework for the application of long-range RNA base pairings in regulating the mutually exclusive splicing of Coleoptera Dscam1.

Antisense RNA-mediated transcriptional attenuation is the mode of replication control of several plasmids, among them pIP501. This mechanism implies that the repR mRNAs can fold into two mutually exclusive structures. The formation of one of these structures is induced by binding of the antisense RNA and results in premature termination. Since the fate of the nascent mRNA transcripts depends on the binding rate of the antisense RNA to its target, the control is kinetic. We have studied the antisense RNA, RNAIII, and target RNA, RNAII, whose interaction determines the replication frequency of plasmid pIP501. RNA secondary structures were analyzed using structure-specific RNases. RNA binding was studied in vitro with normal size and truncated RNAIII species. An in vitro single-round attenuation assay was developed that permits qualitative and quantitative assessment of inhibition by RNAIII. The effect of varying concentrations of RNAIII species on attenuation was tested and inhibition rate constants were calculated. The inhibition rate constants were at least 10 times higher than the pairing rate constants. Thus, steps preceding stable RNA duplex formation are sufficient to induce RNAIII-dependent termination of nascent RNAII transcripts.

Mutually Exclusive Splicing of the Insect Dscam Pre-mRNA Directed by Competing Intronic RNA Secondary Structures

The Drosophila melanogaster gene Dscam1 potentially generates 38,016 distinct isoforms via mutually exclusive splicing, which are required for both nervous and immune functions. However, the mechanism underlying splicing regulation remains obscure. Here we show apparent evolutionary signatures characteristic of competing RNA secondary structures in exon clusters 6 and 9 of Dscam1 in the two midge species (Belgica antarctica and Clunio marinus). Surprisingly, midge Dscam1 encodes only ∼6000 different isoforms through mutually exclusive splicing. Strikingly, the docking site of the exon 6 cluster is conserved in almost all insects and crustaceans but is specific in the midge; however, the docking site-selector base-pairings are conserved. Moreover, the docking site is complementary to all predicted selector sequences downstream from every variable exon 9 of the midge Dscam1, which is in accordance with the broad spectrum of their isoform expression. This suggests that these cis-elements mainly function through the formation of long-range base-pairings. This study provides a vital insight into the evolution and mechanism of Dscam1 alternative splicing.

Polyploid species represent a challenge for both cytogenetic and genomic studies due to their high chromosome numbers and the morphological similarity between their paralogous chromosomes. This paper describes the use of low-coverage high-throughput sequencing to identify the 14 most abundant tandemly arranged repetitive elements in the paleotetraploid genome of the crucian carp (Carassius carassius, 2n = 100). These repetitive elements were then used for molecular cytogenetic studies of a closely related functionally triploid form of the Prussian carp (Carassius gibelio, 3n = 150 + Bs) and a relatively distant diploid species, the tench (Tinca tinca, 2n = 48). According to their distribution on the chromosomes of the 3 aforementioned species, the repetitive elements here identified can be divided into 5 groups: (1) those specific to a single genomic locus in both Carassius species, despite the recent carp-specific genome duplication; (2) those located in a single genomic locus of T. tinca, but amplified in one or both Carassius species; (3) those massively amplified in the B chromosomes of C. gibelio; (4) those located in a single locus in C. gibelio, but amplified in many blocks in C. carassius; and (5) those located in multiple pericentromeric loci in both Carassius species. Our data indicate that some of the repetitive elements are highly conserved in cyprinoid species and may serve as good cytogenetic and genomic markers for discriminating paralogous chromosomes, while others are evolutionarily recent, and their amplification may be related to the last whole-genome duplication event.

Drosophila melanogaster Dscam1 encodes 38,016 isoforms via mutually exclusive splicing; however, the regulatory mechanism behind this is not fully understood. Here, we found a set of hidden RNA secondary structures that balance the stochastic choice of Dscam1 splice variants (designated balancer RNA secondary structures). In vivo mutational analyses revealed the dual function of these balancer interactions in driving the stochastic choice of splice variants, through enhancement of the inclusion of distal exon 6s by cooperating with docking site–selector pairing to form a stronger multidomain pre-mRNA structure and through simultaneous repression of the inclusion of proximal exon 6s by antagonizing their docking site–selector pairings. Thus, we provide an elegant molecular model based on competition and cooperation between two sets of docking site–selector and balancer pairings, which counteracts the “first-come, first-served” principle. Our findings provide conceptual and mechanistic insight into the dynamics and functions of long-range RNA secondary structures.

Drosophila Dscam1 (Down Syndrome Cell Adhesion Molecules) and vertebrate clustered protocadherins (Pcdhs) are two classic examples of the extraordinary isoform diversity from a single genomic locus. Dscam1 encodes 38,016 distinct isoforms via mutually exclusive splicing in D. melanogaster, while the vertebrate clustered Pcdhs utilize alternative promoters to generate isoform diversity. Here we reveal a shortened Dscam gene family with tandemly arrayed 5′ cassettes in Chelicerata. These cassette repeats generally comprise two or four exons, corresponding to variable Immunoglobulin 7 (Ig7) or Ig7–8 domains of Drosophila Dscam1. Furthermore, extraordinary isoform diversity has been generated through a combination of alternating promoter and alternative splicing. These sDscams have a high sequence similarity with Drosophila Dscam1, and share striking organizational resemblance to the 5′ variable regions of vertebrate clustered Pcdhs. Hence, our findings have important implications for understanding the functional similarities between Drosophila Dscam1 and vertebrate Pcdhs, and may provide further mechanistic insights into the regulation of isoform diversity.

A single mouse genomic locus encodes proteins catalyzing three steps of purine synthesis, glycinamide ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide synthetase (AIRS), and glycinamide ribonucleotide formyltransferase (GART). This gene has 22 exons and spans 28 kilobases. The existence of a second genetic locus and closely related pseudogenes was ruled out by Southern analysis. Mouse tissues express two related classes of messages encoded by this single locus: a trifunctional GARS-AIRS-GART mRNA and a monofunctional GARS mRNA. These transcripts used the same set of multiple transcriptional start sites, and both used the same first 10 exons. CCAAT and TATA elements were not found for this locus. Exon 11, which represented the last coding sequence of the GARS domain, was differentially utilized for the two messages. The trifunctional mRNA was generated by splicing exon 11 to exon 12, the first coding sequence for the AIRS domain with subsequent use of a polyadenylation signal at the end of exon 22. Genomic sequence corresponding to the 3'-UTR of the monofunctional GARS mRNA was contiguous with exon 11, so that the smaller message arose from the recognition of one of the multiple polyadenylation signals present within the intron between exons 11 and 12. Hence, polyadenylation of the primary transcript at a position corresponding to an intron of the genomic locus was responsible for the generation of the monofunctional GARS class of mRNAs. This utilization of an intronic polyadenylation site without alternative exon usage is comparable to the mechanism whereby both secreted and membrane-bound forms of the immunoglobulin mu heavy chain are made from a single genetic locus.

Sirtuin 5 (SIRT5) is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, belonging to the silent information regulator 2 (Sir2) family of sirtuin histone deacetylases (sirtuins). The yeast Sir2 protein and its mammalian derivatives are important in epigenetic gene silencing, DNA repair and recombination, cell cycle, microtubule organization and in the regulation of aging. In mammals, 7 sirtuin isoforms have been identified to date of which three (SIRT3, SIRT4 and SIRT5) are localized in the mitochondria, which serve as the center of energy management and the initiation of cellular apoptosis. In the study presented herein, we report the genomic organization and chromosomal localization of the murine sirt5 gene. We have isolated and characterized the murine sirt5 genomic sequence, which spans a region of 24,449 bp and which has one single genomic locus. The murine sirt5 gene consists of 8 exons and encodes a 310-aa protein with a predictive mo-lecular weight of 34.1 kDa and an isoelectric point of 8.90. For the murine sirt5 gene only one single genomic locus has been identified. The gene has been localized to mouse chromosome 13A4 and is flanked by STS-marker 164522 (synonymous WI MRC-RH: 506859).

Mutually exclusive splicing is an important means of increasing the protein repertoire, by which the Down's syndrome cell adhesion molecule (Dscam) gene potentially generates 38,016 different isoforms in Drosophila melanogaster. However, the regulatory mechanisms remain obscure due to the complexity of the Dscam exon cluster. Here, we reveal a molecular model for the regulation of the mutually exclusive splicing of the serpent pre-mRNA based on competition between upstream and downstream RNA pairings. Such dual RNA pairings confer fine tuning of the inclusion of alternative exons. Moreover, we demonstrate that the splicing outcome of alternative exons is mediated in relative pairing strength-correlated mode. Combined comparative genomics analysis and experimental evidence revealed similar bidirectional structural architectures in exon clusters 4 and 9 of the Dscam gene. Our findings provide a novel mechanistic framework for the regulation of mutually exclusive splicing and may offer potentially applicable insights into long-range RNA–RNA interactions in gene regulatory networks.

An improved set of infrared calibration spectra for the determination of G X C and A X U base pairs leads to 32 +/- 3 G X C (+ G X U) and 4 +/- 1 A X U base pairs for Thermus thermophilus 5S RNA in the presence and absence of Mg2+. These results give further support for the consensus secondary structure of 5S RNA recently proposed by several groups. T. thermophilus 5S RNA shows, in the presence of Mg2+, a distinct two-step thermal melting of its ordered structure. Based on new data about the stacking dependence of infrared intensities of unpaired ribonucleotides the spectral changes of the low-temperature transition should be explained by melting of stacked arrangements of unpaired bases and/or non-standard base pairs. Striking is the reduction in A stacking, which is not related to the melting of A X U base pairs, indicating the importance of the mostly conserved unpaired adenines for the Mg2+ stabilized higher-order structures especially within internal loops of 5S RNA.

Single genomic loci are often related to specific cellular functions, genetic diseases, or pathogenic infections. Visualization of single genomic loci in live human cells is currently of great interest, yet it remains challenging. Here, we describe a strategy for live cell imaging of single genomic loci by combining transcription activator-like effectors (TALEs) with a quantum dot labelling technique. We design and select a pair of TALEs that specifically target HIV-1 proviral DNA sequences, and use bioorthogonal ligation reactions to label them with different colour quantum dots (QDs). These QD-labelled TALEs are able to enter the cell nucleus to provide fluorescent signals to identify single gene loci. Based on the co-localization of the pair of different coloured QD-labelled TALEs, we determine and map single-copy HIV-1 provirus loci in human chromosomes in live host cells.

Biotin proximity labeling has largely extended the toolbox of mass spectrometry-based interactomics. To date, BirA, engineered BirA variants, or other biotinylating enzymes have been widely applied to characterize protein interactions. By implementing chromatin purification-based methods the genome-wide interactome of proteins can be defined. However, acquiring a high-resolution interactome of a single genomic locus preferably by multiplexed measurements of several distinct genomic loci in parallel remains challenging. We recently developed CasID, a novel approach where the catalytically inactive Cas9 (dCas9) is coupled to the promiscuous biotin ligase BirA (BirA∗). With CasID, first the local proteome at repetitive telomeric, major satellite, and minor satellite regions was determined. With more efficient biotin ligases and sensitive mass spectrometry, others have successfully identified the chromatin composition at even smaller genomic, non-repetitive regions of a few hundred base pairs in length. Here, we summarize the most recent developments towards interactomics at a single genomic locus and provide a step-by-step protocol based on the CasID approach.