Association of microRNAs with Argonaute proteins in the malaria mosquito Anopheles gambiae after blood ingestion (original) (raw)

Drastic changes in gene expression occur after adult female mosquitoes take a blood meal and use the nutrients for egg maturation. A growing body of evidence indicates that microRNAs (miRNAs) contribute to this tightly controlled tissue-and stage-specific gene expression. To investigate the role of miRNAs, we monitored miRNA expression in the mosquito Anopheles gambiae during the 72-h period immediately after blood feeding. We also measured the association of miRNAs with Argonaute 1 (Ago1) and Argonaute 2 (Ago2) to assess the functional status of individual miRNA species. Overall, 173 mature miRNAs were precipitated with Ago1 and Ago2, including 12 new miRNAs, the orthologs of which are found thus far only in other Anopheles species. Ago1 is the predominant carrier of miRNAs in Anopheles gambiae. The abundance and Ago loading of most of the mature miRNAs were relatively stable after blood ingestion. However, miRNAs of the miR-309/286/2944 cluster were considerably upregulated after blood feeding. Injection of the specific antagomir for miR-309 resulted in smaller developing oocytes and ultimately fewer eggs. In addition, the Ago association of some miRNAs was not proportional to their cellular abundance, suggesting that integration of miRNAs into the Ago complexes is regulated by additional mechanisms. The Anopheles gambiae mosquito is the major vector of human malaria in Africa. Malaria is caused by protozoan parasites belonging to the genus Plasmodium and is spread between hosts by the bites of parasite-infected female mosquitoes. Female adults of An. gambiae must obtain blood from vertebrates to acquire nutrients for egg production. The female mosquitoes digest a protein-rich blood meal, greater than their original body weight, within 48 h after ingestion and use the nutrients to synthesize egg yolk proteins. Substantial physiological changes occur after blood ingestion in mosquitoes, and over 50% of all genes show significant variation in transcript accumulation 1. Recent studies suggest that blood digestion and egg production in An. gambiae and other mosquito species are also regulated by mosquito microRNAs (miRNAs) 2. Expression of some miRNAs is considerably altered after blood ingestion in An. gambiae 3 , An. stephensi 4 , Aedes aegypti 5 , and Aedes albopictus 6. Functional studies in Ae. aegypti have shown that perturbation of individual miRNAs in female mosquitoes leads to defects in blood digestion and egg production 7-10. miRNAs are small endogenous non-coding RNAs that play important roles in post-transcriptional gene regulation in plants and animals 11. Most miRNAs are transcribed by RNA polymerase II as primary-miRNA (pri-miRNA) transcripts. The canonical miRNA pathway converts the pri-miRNA hairpins into ∼22-nucleotide (nt) mature duplex miRNAs via consecutive cleavages by two RNase III enzymes, Drosha and Dicer, with the help of some double-stranded RNA-binding proteins 12. The two strands of the mature miRNA duplex are denoted by the suffix-5p or-3p, depending on whether they originate from the 5′ or 3′ end of the pri-miRNA. Only one strand of the miRNA duplex is usually loaded onto an Argonaute (Ago) protein as part of the miRNA-induced silencing complex (miRISC). The strand in miRISC is known as the guide strand; the other strand (the passenger strand) is excluded and subsequently degraded 12. The guide strand directs the miRISC to messenger RNA