Investigation of the functional role of the conserved sequence at the 5’-end of the fourth intron of the mod(mdg4) gene in trans-splicing in Drosophila melanogaster

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Abstract

Alternative splicing is an important mechanism that provides genetic diversity of proteins. Unique loci have been identified in Drosophila melanogaster, where mRNA diversity arises as a result of trans-splicing — a process in which exons from different pre-mRNAs are joined together. The trans-splicing in the mod(mdg4) locus, which encodes more than 31 isoforms, has been studied in detail. Important elements for this process include previously described conserved sequences in the fourth intron. The aim of this study is to further characterize the conserved motifs of the fourth intron, specifically the element at the 5’-end of the intron. Using model transgenic lines, it has been shown that introduced changes in the sequence of the studied element lead to a disruption of trans-splicing. In contrast, similar changes in the endogenous locus did not result in a disruption of trans-splicing. Thus, the conserved element plays a role in trans-splicing but is not critical.

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About the authors

Iu. V. Soldatova

Institute of Gene Biology Russian Academy of Sciences

Author for correspondence.
Email: me@mtih.me
Russian Federation, Moscow

O. Beginyazova

Institute of Gene Biology Russian Academy of Sciences

Email: me@mtih.me
Russian Federation, Moscow

P. G. Georgiev

Institute of Gene Biology Russian Academy of Sciences

Email: me@mtih.me

Academician of the RAS

Russian Federation, Moscow

M. V. Tikhonov

Institute of Gene Biology Russian Academy of Sciences

Email: me@mtih.me
Russian Federation, Moscow

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Supplementary files

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2. Fig. 1. (A) Structure of the mod(mdg4) locus in Drosophila melanogaster. Annotated promoters are indicated by arrows. Common exons present in all isoforms are shown in green. The splice donor site involved in trans-splicing is located after the fourth common exon (indicated by the red semicircle). Alternative exons unique to a particular isoform are shown in orange (on the same DNA strand) and purple (on the opposite strand). (B) Alignment of the 5' splice site of the fourth intron and its adjacent sequences with homologous regions from other species. Sequences from the family Drosophilidae and the flies Musca_domestica, Glossina morsitans are shown. (B) Logo representation of the 5'CC motif of all Drosophila melanogaster introns, obtained for the current annotation of the D. melanogaster genome [16] and visualized using WebLogo [17]. (D) Changes made to the sequence at the 5' end of the intron.

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3. Fig. 2. (A) Schematic of a model system designed to study the functional role of the conserved region at the 5' end of intron 4 of the mod(mdg4) gene. The “donor construct” (left) contains the mod(mdg4) promoter (arrow), the coding region consisting of four common exons (green rectangles), the IRES, and intron 4 (5'CC is the red semicircle, the transcribed part of the intron is the red gradient). The “acceptor construct” (right) contains a bidirectional promoter of the T and K isoforms (arrows) and outrons (gray) with 3'CC (red semicircle) of the corresponding isoforms. Transcription results in the formation of a donor and two acceptor transcripts. Trans-splicing produces two types of mRNA consisting of the 5' part of the donor transcript and the 3' part of the acceptor transcripts. The annealing sites of the primers for the analysis of trans-splicing efficiency are indicated by arrows under the corresponding transcripts. (B) Trans-splicing efficiency in donor/acceptor heterozygotes was assessed by RT-qPCR. Trans-splicing efficiency was estimated by the ratio of the amounts of amplicons from the junction of the fourth exon and the marker gene to two housekeeping genes (Vha100-1, CG9067). Each combination of trans-heterozygotes was analyzed in at least three replicates. Error bars represent standard deviations (n ​​= 3). Asterisks denote significance levels: ***P < 0.001.

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4. Fig. 3. (A) Schematic representation of the modification of the endogenous mod(mdg4) locus. CRISPR/Cas9 is used to make a cut in intron-4 (scissors icon). A vector with a mutation in one of the arms was used as a template for recombination (sequence changes are shown in red). For the selection of transformants, the construct contained the fluorescent marker mCherry surrounded by loxP sites, due to which this gene was removed by Cre recombinase. Two types of transformants were observed: with a mutation (mut-loxP) or with the wild-type sequence (wt-loxP). (B) Evaluation of the trans-splicing efficiency in wild-type flies and in homozygous lines with a loxP site and with a mutant version of the 5' sequence and a loxP site. Trans-splicing efficiency was estimated by the ratio of amplicons from the junction of the fourth exon and alternative exons T, K, Z to two housekeeping genes (Vha100-1, CG9067). Measurements for each line were repeated at least three times. Error bars represent standard deviations (n ​​=3).

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