Single Nucleotide Polymorphism (SNP) in splice sites and prediction for the doublesex (dsx) gene alternative splicing in the malaria vector Anopheles gambiae.

doi:10.21203/rs.3.rs-118845/v1

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Single Nucleotide Polymorphism (SNP) in splice sites and prediction for the doublesex (dsx) gene alternative splicing in the malaria vector Anopheles gambiae.

https://doi.org/10.21203/rs.3.rs-118845/v1

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Background: Malaria burden continues to be significant in tropical regions, and conventional vector control methods are faced with challenges such as insecticide resistance. To overcome these challenges, additional vector control interventions are vital and include modern genetic approaches as well as classical methods like the sterile insect technique (SIT). In the major human malaria vector Anopheles gambiae, a candidate gene favourable for sterility induction is the doublesex (dsx) gene, encoding somatic sexually dimorphic traits in mosquitoes. However, the mechanism that regulates the expression of this gene in anopheline mosquitoes is poorly understood. This study aims to screen the An. gambiae dsx gene for single-nucleotide polymorphisms (SNPs) that could be critical to its alternative splicing.

Result: Variant annotation data from Ag1000G project phase 2 was analysed, in order to identify splice-relevant SNPs within acceptor and donor splice sites of the An. gambiae dsx gene (Agdsx). SNPs were found in both donor and acceptor site of the Agdsx. No splice-relevant SNPs were identified in female-specific intron 4 acceptor site and the corresponding region in males. Two SNPs (rs48712947, rs48712962) were found in female-specific donor site of exon 5. They were not specific to either males or females. as the rs48712947 was found in female mosquitoes from Cameroon, and in both males and females from Burkina Faso. In the other splice sites, the intron 3 acceptor site carried the greatest abundance of SNPs.

Conclusion: There were no gender association between the identified SNPs and the random distribution of these SNPs in mosquito populations. The SNPs in Agdsx splice sites are not critical for the alternative splicing. Other molecular mechanisms should be considered and investigated.

Epigenetics & Genomics

SNP

alternative splicing

dsx gene

Anopheles gambiae

malaria

Malaria is a vector-borne infectious disease caused by the protozoan parasite belonging to the Plasmodium genus (1). The transmission occurs among humans through the bite of the female Anopheles mosquito. This disease is among the top ten causes of death in low-income countries (2) and continues to take a heavy toll on communities, especially in Africa regions. The malaria transmission cycle involves four major elements: the host (human), the parasite, the vector and the environment (3). In the absence of effective vaccine or sustainable treatment options, vector control is the cornerstone of malaria management and is based on prevention of human-host contact and reduction in vector population density (1, 4). The traditional vector control strategies rely on long-lasting insecticidal net (LLIN) distribution and indoor residual sprays (IRS) which have contributed to the decreasing malaria cases and mortality (5, 6). However, vector resistance against the existing insecticides is increasing in natural mosquito populations (7–9).

In the last decade, scientific advances in additional tools for vector control include technologies such as cytoplasmic incompatibility with the use of Wolbachia infection (10); repressible dominant lethal systems in Aedes aegypti (11, 12); Y-chromosome shredding gene drive (13); and the genetic sterilisation of Anopheles sp., known as Sterile Insect Techniques (SIT) (14). The latter technique, SIT, is based on the repeated, high-density release of radio-sterilized males, through gamma radiation, into the environment in order to compete with wild males for mating with the native female anopheles mosquitoes hindering the production of offspring (14). Indeed, mated females will not produce viable offspring, resulting in reduced population numbers or even elimination of the target species. However, instead of exposing males to a source of radiation, sterility could be induced by genetic modification of the mosquito genome and may improve the effectiveness of classical SIT-based approaches (14).

In An. gambiae, one of the major malaria vectors, population supression strategies are already under investigation by targeting the sex determination genes such as the doublesex (dsx) transcription factor gene (15, 16). Therefore, the Anopheles gambiae doublesex gene (Agdsx) represent a useful candidate gene for genetic manipulation and improvement of the alternative mosquito control technologies. Interest in this gene comes from the fact that it undergoes alternative splicing and result in female and male-specific transcripts necessary for sex determination in this species (17). The use of transgenic tools in anopheline mosquitoes through targeting the dsx gene could improve the sterility induction and genetic sexing which are major requirements for SIT technologies. However, the molecular mechanisms underlying gender determination are highly variable. Though it was demonstrated that Yob1 gene (Y-linked) is one of the determining factors of the male sex (18), the molecular pathways controlling the signal of somatic sexual commitment (dsx splicing and regulation) in An. gambiae are not well understood. The only well-known model of the dsx splicing comes from the fly Drosophila melanogaster sex determination pathway (19). The dsx gene acts as a transcription factor targeting several genes which have mostly sex- and tissue-specific functions in fly (20, 21). Transformer (TRA) and Transformer 2 (TRA2) are the key regulatory factors of the female-specific alternative splicing of dsx pre-mRNA while the absence of TRA lead to the male-specific splicing (20). Both TRA and TRA2 are downstream targets of the Sex lethal gene (Sxl) product (19). Unfortunately, An. gambiae dsx gene (Agdsx) has a different structure suggesting that Agdsx sex-specific splicing event is caused by a mechanism different from that of the D. melanogaster dsx (17, 22).

In mammalian cells, the presence of genetic variations such as single nucleotide polymorphisms (SNPs) within the donor and acceptor splice sites could influence splicing and might lead to changes in normal splicing pattern (23–25). Donor sites (5’-splice site) are defined by GT dinucleotide at the 5’ end of exon-intron border, while AG dinucleotide defined acceptor sites (3’-splice site) at the 3’ end of intron-exon border (26). Thus, we hypothesize that SNPs could occur in the Agdsx acceptor and donor splice sites that might result in the splice variation With this in mind, the current report seeks to screen Agdsx for single-nucleotide polymorphisms (SNPs) that could be associated with alternative splicing.

Identification of An. gambiae dsx gene (Agdsx) donor and acceptor splice sites sequence.

Agdsx is located in the band 17C of the chromosome 2R (2R: 48703664–48788460) on the reverse strand. The gene is 84.8 kb long and encodes the male and female-specific transcript. The male transcript (6975 bp) is shorter than that of female (8667 bp). The difference between the two sex-specific transcripts is due to the alternative splicing of exon 5. This latter is a cassette exon, which is retained in female and skipped in male transcript. The whole sequence of female specific exon 5 is included in male intron 4 region and is spliced out. This gene structure causes a shift in intron/exon number in male. Thus, male and female have common and specific splice sites.

Male and female mosquitoes share exon 1, 2, 3, 4 and 6 donor splice sites while exon 5 donor site is specific to female (Table 2). Similarly, both sexes share intron 1, 2, 3, and 6 acceptor sites. Male intron 4 and female intron 5 share the same 3’ end as the female exon 5 is included in the male intron 4 sequence. However females have the intron 4 specific acceptor site (Table 2).

Table 2

Splice donor and acceptor sites within the double sex (*dsx*) gene *of Anopheles gambiae*.
Splice donor sites
Sex	Exon	Size	Exon position		Splice site sequence	Site position
			Start	End		Start	End
Male/Female	1	1415	48788460	48787046	tatttg/gtaagtaaatatgcaa	48787051	48787030
Male/Female	2	1445	48785629	48784185	TGGGAG/gtaagtacgatcatgc	48784190	48784169
Male/Female	3	45	48747737	48747693	TACCTG/gtaagtaaatataatt	48747698	48747677
Male/Female	4	135	48715295	48715161	ACGAAG/gtaagctggcgatgat	48715166	48715145
Female	5	1692	48714648	48712957	cagaag/gtatggtaagacggcc	48712962	48712941
Male/Female	6	1267	48712794	48711528	aaaaag/gtaagtgtgggtagta	48711533	48711512
Male/Female	7	2668	48706331	48703664	None
Splice acceptor sites
Sex	Intron	Size	Intron position		Splice site sequence	Site position
			Start	End		Start	End
Male/Female	1	1416	48787045	48785630	gtacgtttgattgcag/atctcc	48785645	48785624
Male/Female	2	36447	48784184	48747738	ttgctctccttttcag/CTACTC	48747753	48747732
Male/Female	3	32397	48747692	48715296	ttccgccccgtttcag/ACGACG	48715311	48715290
Female	4	512	48715160	48714649	tttatgtttaacacag/GTCAAG	48714664	48714643
Male	4	2366	48715160	48712795	tgtaacccccaaaaag/gtaaac	48712810	48712789
Female	5	162	48712956	48712795	tgtaacccccaaaaag/gtaaac	48712810	48712789
Male/Female	6	5196	48711527	48706332	cgcttcctcaaaatag/atcgat	48706347	48706326
Splice site sequences are given in 5’⎝3’ direction on the reverse strand. Exonic coding sequences are shown in uppercase letters, and non-coding regions are in lowercase letters. The 12 bp preceding the 3’ splice-acceptor site (NYag) is indicated, where Y = T or C and N = any nucleotide.
SNPs in female-specific intron 4 acceptor and exon 5 donor splice sites.

Along the Agdsx gene, 17196 polymorphic sites were identified. The nucleotide diversity is similar between male and female mosquitoes (Fig. 1). The potential splice-relevant SNPs that could trigger the female-specific exon 5 skipping should be in the intron 4 acceptor and exon 5 donor sites. However, there was no SNP in acceptor sequence of female-specific intron 4 nor in the male corresponding region (Fig. 2). However in female specific exon 5 donor site, two SNPs (rs48712947, rs48712962) were found. Nevertheless, they were not specific to female as the rs48712947 was found in Cameroon female mosquitoes and in both males and females from Burkina Faso (Fig. 3). The rs48712962 was absent in male mosquito population, while it was found only in Cameroon females. The minor allele frequencies (MAF) of both SNPs identified were very low in each population. The MAF of rs48712947 and rs48712962 were less than 1% in each female population and only 2% of Burkina Faso male carried the rs48712947. Moreover, none of these SNPs were associated with the sex phenotype (rs48712947: p = 0.32; rs48712962: p = 0.68).

SNPs in other splice sites of Agdsx.

The other splice sites were also examined for identification of sex-specific SNPs (see additional Figs. 1–3). No SNP was found in the shared exon 1 donor, introns 1 and 6 acceptor sites. The highest number of SNPs (07) was found in the common intron 3 acceptor site sequence (rs48715291, rs48715294, rs48715302, rs48715306, rs48715307, rs48715308, rs48715309) (Fig. 4). However, each of these SNPs occurred in non-specific manner in both male and female populations with variable minor allele frequencies.

The An. gambiae doublesex (Agdsx) gene is a candidate gene of interest for sterile insect technique (SIT), as a candidate for genetic modifications (15, 16). The translation and the success of using dsx in SIT methodology require a clearer understanding of the genetic bases of the sex determination pathway. This study screened the Agdsx donor and acceptor splice sites for identification of splice-relevant SNPs.

According to the D. melanogaster model (19), the alternative splicing of Agdsx gene is governed by exon 5 skipping in male mosquitoes (17) suggesting a silencing mechanism of the female-specific splice sites recognition (intron 4 acceptor and exon 5 donor sites) by the splicing machinery in male. Such silencing mechanism could be due to changes in splice site sequence. However, female-specific intron 4 acceptor site sequence is present within male intron 4 and no SNP was found in this sequence in both sex. The SNPs rs48712947 and rs48712962 identified in female-specific exon 5 donor site were neither splice-relevant nor sex-specific. They appeared only is two mosquito populations (Burkina Faso and Cameroon) over the eight populations considered. In each population where these SNPs have been identified, they appeared in very few individuals, less than 1% in females and no more than 2% in males. These observations suggest that the Agdsx cassette exon 5 was not associated with changes in splice site patterns due to the presence of SNPs. The presence of SNPs in the other splice sites had also different distribution and were non-specific to the gender of the mosquitoes.

Another factor for exon skipping is the pyrimidine content of the polypyrimidine tract in acceptor splice sequence. Indeed a poor polypyrimidine tract cause a shift of the splicing machinery to the next acceptor site, leading to the skipping as the case of exon 4 skipping in male Drosophila (19). In Anopheles gambiae the number of pyrimidine (8) in the 12 bp preceding the acceptor site pattern (acag) (Table 2) in the female-specific intron 4 is the same in the male corresponding region. The same number of pyrimidines in this acceptor sequence was reported by Scali et al. (17). Furthermore, the authors found that this number did not differ from the consensus number of pyrimidines (8.69) in An. gambiae splice acceptor sites, and concluded that the intron 4 site may not be a weak acceptor site (17). The Drosophila dsx splicing regulation involves the products of transformer (Tra) and transformer 2 (Tra2) genes (19). Although ortholog of Drosophila Tra2 is present in An. gambiae (accession number: AGAP006798), no ortholog of Tra was found in An. gambiae genome (www.flybase.org). This suggests that Tra may be missing in An. gambiae or its splicing regulatory function could be ensured by another gene. Overall, these findings add another evidence that other mechanisms underlie the alternative splicing in An. gambiae and open perspective for further investigation on the molecular mechanisms of Agdsx splicing.

It was known that the regulation of alternative splicing evolved tans-acting splicing factors, such as serine-arginine-rich (SR) family proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs) that bind to the auxiliary silencer and enhancer cis-element (ESE: exonic splicing enhancers; ESI: exonic splicing silencers; ISE: intronic splicing enhancers; ISI: intronic splicing silencers) (27–29). Similar regulatory cis-elements were found in Drosophila melanogaster female-specific exon and putative homologs were identified in An. gambiae female-specific exon 5 (17). Therefore, further molecular analysis are needed toward characterizing these regulatory sequence and their binding trans-factors in order to underpin the somatic sex determination in An. gambiae.

Moreover, the epigenetic system was also reported to regulate the alternative splicing in mammalian and other insects cells. Indeed, it was demonstrated that changes in DNA cytosine methylation on the gene body in honey bees may lead to alternative splicing (30–32). Also histone post-translational modifications (PTMs) such as lysine acetylation and methylation were associated to the alternative splicing event (33–35). Consequently, similar mechanisms could happen in the malaria vector An. gambiae to regulate gene alternative splicing. However, no significant DNA methylation was reported in Diptera including An. gambiae (36, 37). Then, the only epigenetic modifications that could be linked to the alternative splicing in this species are histone PTMs. Indeed, the methylation and acetylation of lysines 4, 9 and 29 of histone H3 were reported in An. gambiae (38, 39). Then, it will be interesting to evaluate whether such histone modifications enrichment in Agdsx between male and female mosquitoes could be critical for dsx alternative splicing.

Sustainable vector control strategies will rely on the integrated use of chemicals and biological vector control. Given the potential of the Anopheles gambiae dsx (Agdsx) gene for sterile insect technique (SIT), the understanding of mechanisms of it regulation could help to improve the tools engineering targeting this locus. SNPs were identified within the Agdsx and their putative association with the dsx alternative splicing was analysed. No splice-relevant SNP was found in the specific male and female splice site. The SNPs were distributed in few proportion of individuals in the populations where they were identified. With the advances in genetic biotechnology, other mechanisms remain to be explored for providing solid background on somatic sexual fate determination in Anopheles gambiae. This will pave the way to find new biochemical and genetics target for malaria vector control.

Sequence data and mosquito samples

Genomic sequences used in this study came from the Anopheles 1000 genomes (Ag1000G) project phase 2 released in 2017 (22). The SNP annotation was downloaded (ag1000g.phase2.ar1.variants.pass.2R.vcf.gz, November 11, 2019) from the Malaria Genomic Epidemiology Network (MalariaGEN) website. This file contain all SNPs identified in mosquitoes whole genome and that pass the variant filtering process described by (22). Only Anopheles gambiae samples were considered in our study. These mosquito samples were collected from natural populations from 2002 to 2012 in eight African countries (Table 1). The reference sequence of Agdsx (AGAP004050) was also downloaded from Vectorbase website.

Table 1

Sampling locations of *An. gambiae* mosquitoes from Ag1000G phase 2 project.
Country	Site	Year	Geographic coordinate		Number of species
Country	Site	Year	Latitude	Longitude	Number of species
Burkina Faso	Bana	2012	11.2330	-4.4720	20
	Pala	2012	11.1500	-4.2350	46
	Souroukoudinga	2012	11.2350	-4.5350	26
Cameroon	Daiguene	2009	4.7770	13.8440	96
	Gado Badzere	2009	5.7470	14.4420	73
	Mayos	2009	4.3410	13.5580	105
	Zembe Borongo	2009	5.7470	14.4420	23
Equatorial Guinea	Bioko	2002	3.7000	8.7000	9
France (Mayotte)	Bouyouni	2011	-12.7378	45.1417	1
	Combani	2011	-12.7787	45.1429	5
	Karihani Lake	2011	-12.7965	45.1217	3
	Mont Benara	2011	-12.8570	45.1552	2
	Mtsamboro Forest Reserve	2011	-12.7027	45.0811	1
	Mtsanga Charifou	2011	-12.9907	45.1557	8
	Sada	2011	-12.8521	45.1039	4
Gabon	Libreville	2000	0.3840	9.4550	69
Ghana	Madina	2012	5.6685	-0.2193	12
Guinea	Koraboh	2012	9.2500	-9.9170	22
Guinea	Koundara	2012	8.5000	-9.4170	18
Uganda	Tororo (Nagongera)	2012	0.7700	34.0260	112

Sequence analysis and SNP identification

From the Agdsx reference sequence, the list of genomic positions of donor and acceptor sites was extracted. VCFtools version 0.1.15 (40) was used to extract the SNPs within the genomic region corresponding to Agdsx sequence from the SNPs annotation file. The polymorphic nucleotides were then identified within the splice sequences, in comparison to the reference sequence. SNPs were then visualized using TASSEL version 5.2.63 software (41). The genomic position of the acceptor sites was used to select SNPs in the last 12 nucleotides of an intron preceding the 3’ splice pattern NYAG and in the first 6 nucleotides of an exon. In donor splice sites SNPs were identified within in the last 6 nucleotides of an exon and the first 16 nucleotide in an intron. The SNPs association to the sex phenotype (male or female) was evaluated by running the association analysis using the general linear model (GLM) function in TASSEL. The averages nucleotide diversity at the dsx locus between male and female was calculated using scikit-allel version 1.2.1 (42) in order to determine whether SNPs density at the dsx locus is different between the two genders.

Agdsx

Anopheles gambiae doublesex gene

dsx

doublesex gene

ESE

Exonic Splicing Enhancers

ESI

Exonic Splicing Silencers

hnRNPs

heterogeneous nuclear ribonucleoproteins

ISE

Intronic Splicing Enhancers

ISI

Intronic Splicing Silencers

PTMs

post-translational modifications

SIT

Sterile Insect Techniques

SNP

Single Nucleotide Polymorphism

Sxl

Sex lethal gene

TRA

Transformer transcription factor

TRA2

Transformer 2 transcription factor

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and materials

The datasets generated and/or analysed during the current study are available from Ag1000G phase 2 via the MalariaGEN website (ftp://ngs.sanger.ac.uk/production/ag1000g/phase2/AR1/variation/main/vcf/pass_variants/)

Competing interests

The authors declare that they have no competing interests

Funding

Not applicable

Authors' contributions

HS retrieved data and extracted data corresponding to the genomic region of interest. OD analysed data and wrote the manuscript. LD contributed to the critical review of the draft manuscript. All authors read and approved the final manuscript.

Acknowledgements

The authors are grateful to Dr. Luisa Nardini, Dr. Lizette Koekemoer and Dr David Weetman for their helpful comments on this manuscript. The authors also acknowledge Wellcome Trust for financial support to LSD (grant 109917/Z/15/Z).

Authors' information (optional)

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Single Nucleotide Polymorphism (SNP) in splice sites and prediction for the doublesex (dsx) gene alternative splicing in the malaria vector Anopheles gambiae.

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Sequence analysis and SNP identification

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