Elav-mediated exon skipping and alternative polyadenylation of the Dscam1 gene is required for axon outgrowth

SummaryMany metazoan genes express alternative long 3′ UTR isoforms in the nervous system, but their functions remain largely unclear. In Drosophila melanogaster, the Dscam1 gene generates short and long (Dscam1-L) 3′ UTR isoforms due to alternative polyadenylation (APA). Here, we found that the RNA-binding protein Embryonic Lethal Abnormal Visual System (Elav) impacts Dscam1 biogenesis at two levels, including regulation of long 3′ UTR biogenesis and skipping of an upstream exon (exon 19). MinION long-read sequencing confirmed the connectivity of this alternative splicing event to the long 3′ UTR. Knockdown or CRISPR deletion of Dscam1-L impaired axon growth in Drosophila. The Dscam1 long 3′ UTR was found to be required for correct Elav-mediated skipping of exon 19. Elav thus co-regulates APA and alternative splicing to generate specific Dscam1 transcripts that are essential for neural development. This coupling of APA to alternative splicing might represent a new class of regulated RNA processing. Graphical AbstractHighlightsElav regulates Dscam1 long 3′ UTR (Dscam1-L) biogenesisLong-read sequencing reveals connectivity of long 3′ UTR to skipping of upstream exon 19Loss of Dscam1-L impairs axon outgrowthDscam1 long 3′ UTR is required for correct splicing of exon 19

Self-Splicing of the Bacteriophage T4 Group I Introns Requires Efficient Translation of the Pre-mRNA In Vivo and Correlates with the Growth State of the Infected Bacterium

ABSTRACT Bacteriophage T4 contains three self-splicing group I introns in genes in de novo deoxyribonucleotide biosynthesis (in td, coding for thymidylate synthase and in nrdB and nrdD, coding for ribonucleotide reductase). Their presence in these genes has fueled speculations that the introns are retained within the phage genome due to a possible regulatory role in the control of de novo deoxyribonucleotide synthesis. To study whether sequences in the upstream exon interfere with proper intron folding and splicing, we inhibited translation in T4-infected bacteria as well as in bacteria containing recombinant plasmids carrying the nrdB intron. Splicing was strongly reduced for all three T4 introns after the addition of chloramphenicol during phage infection, suggesting that the need for translating ribosomes is a general trait for unperturbed splicing. The splicing of the cloned nrdB intron was markedly reduced in the presence of chloramphenicol or when translation was hindered by stop codons inserted in the upstream exon. Several exon regions capable of forming putative interactions with nrdB intron sequences were identified, and the removal or mutation of these exon regions restored splicing efficiency in the absence of translation. Interestingly, splicing of the cloned nrdB intron was also reduced as cells entered stationary phase and splicing of all three introns was reduced upon the T4 infection of stationary-phase bacteria. Our results imply that conditions likely to be frequently encountered by natural phage populations may limit the self-splicing efficiency of group I introns. This is the first time that environmental effects on bacterial growth have been linked to the regulation of splicing of phage introns.

Identification of upstream regulatory regions in the heart-expressed homeobox gene Nkx2-5

Nkx2-5 marks the earliest recognizable cardiac progenitor cells, and is activated in response to inductive signals involved in lineage specification. Nkx2-5 is also expressed in the developing foregut, thyroid, spleen, stomach and tongue. One approach to elucidate the signals involved in cardiogenesis was to examine the transcriptional regulation of early lineage markers such as Nkx2-5. We generated F0 transgenic mice, which carry Nkx2-5 flanking sequences linked to a lacZ reporter gene. We identified multiple regulatory regions located within the proximal 10.7 kb of the Nkx2-5 gene. In addition to a proximal promoter, we identified a second promoter and a novel upstream exon that could participate in the regulation of Nkx2-5 transcription. Although used rarely in normal development, this novel exon could be spliced into the Nkx2-5 coding region in several ways, thereby potentially creating novel Nkx2-5 protein isoforms, whose transcriptional activity is greatly diminished as compared to wild-type Nkx2-5. An enhancer that directs expression in pharynx, spleen, thyroid and stomach was identified within 3.5 kb of exon 1 between the coding exon 1 and the novel upstream exon 1a. Two or more enhancers upstream of exon 1a were capable of driving expression in the cardiac crescent, throughout the myocardium of the early heart tube, then in the outflow tract and right ventricle of the looped heart tube. A negative element was also located upstream of exon1a, which interacted in complex ways with enhancers to direct correct spatial expression. In addition, potential autoregulatory elements can be cooperatively stimulated by Nkx2-5 and GATA-4. Our results demonstrate that a complex suite of interacting regulatory domains regulate Nkx2-5 transcription. Dissection of these elements should reveal essential features of cardiac induction and positive and negative signaling within the cardiac field.

Intron Definition Is Required for Excision of the Minute Virus of Mice Small Intron and Definition of the Upstream Exon

ABSTRACT Alternative splicing of pre-mRNAs plays a critical role in maximizing the coding capacity of the small parvovirus genome. The small-intron region of minute virus of mice (MVM) pre-mRNAs undergoes an unusual pattern of overlapping alternative splicing—using two donors (D1 and D2) and two acceptors (A1 and A2) within a region of 120 nucleotides—that determines the steady-state ratios of the various viral mRNAs. In this report, we show that the determinants that govern excision of the small intron are complex and are also required for efficient definition of the upstream exon. For the MVM small intron in its natural context, the two donors appear to compete for the splicing machinery: the position of D1 favors its usage, while the primary sequence of D2 must be more like the consensus sequence than is D1 to be used efficiently. We have genetically defined the branch points that are used for generation of the major and minor spliced forms and show that recognition of components of the small-intron acceptors is likely to be the dominant determinant in alternative small-intron excision. We have also identified a G-rich intronic enhancer sequence within the small intron that is essential for splicing of the minor form (D2 to A2) but not the major form (D1 to A1) of MVM mRNAs and is required for efficient definition of the upstream NS2-specific exon. In its natural context, the small intron appears to be excised by a mechanism consistent with intron definition. When the MVM small intron is expanded, various parameters of its excision are altered, indicating that critical cis-acting signals are context dependent. Relative use of the donors and acceptors is altered, and the upstream NS2-specific exon is no longer efficiently defined. The fact that definition of the upstream NS2-specific exon can be achieved by the MVM small intron in its natural context, but not when it is expanded, suggests that the multiple determinants that govern definition and excision of the small intron are required, in concert, for upstream exon definition. Our data are consistent with a model in which alternative splicing of the MVM P4-generated pre-mRNAs is governed by a hybrid of intron- and exon-defining mechanisms.