SNPs in human miRNA genes affect biogenesis and function

Abstract

MicroRNAs (miRNAs) are 21-25-nucleotide-long, noncoding RNAs that are involved in translational regulation. Most miRNAs derive from a two-step sequential processing: the generation of pre-miRNA from pri-miRNA by the Drosha/DGCR8 complex in the nucleus, and the generation of mature miRNAs from pre-miRNAs by the Dicer/TRBP complex in the cytoplasm. Sequence variation around the processing sites, and sequence variations in the mature miRNA, especially the seed sequence, may have profound affects on miRNA biogenesis and function. In the context of analyzing the roles of miRNAs in Schizophrenia and Autism, we defined at least 24 human X-linked miRNA variants. Functional assays were developed and performed on these variants. In this study we investigate the affects of single nucleotide polymorphisms (SNPs) on the generation of mature miRNAs and their function, and report that naturally occurring SNPs can impair or enhance miRNA processing as well as alter the sites of processing. Since miRNAs are small functional units, single base changes in both the precursor elements as well as the mature miRNA sequence may drive the evolution of new microRNAs by altering their biological function. Finally, the miRNAs examined in this study are X-linked, suggesting that the mutant alleles could be determinants in the etiology of diseases.

FIGURE 1.

Functional test of miR-502 and miR-502-C/G. (A) Sequences of miRNAs and reporters. Stem–loop sequences of miR-502, miR-502-C/G, and the sequences inserted into the 3′UTR of Rluc for the “si” and “mi” reporters. (B) Cotransfection test results. The knockdown of 5p-si, 5p-mi, and 3p-si reporters is reduced in the mutant, whereas the expression of 3p-mi reporter is the same in the WT and Mutant. Each bar represents the average of at least three independent transfections with duplicate determinations for each construct. Error bars represent the SD. (C) Northern blot results. Blot was hybridized with miR-502-3p probe (right) and a miR-502-5p probe (left). (Lane 1) RNAs from cells transfected with the WT miRNA construct; (lane 2) samples from cells transfected with the Mutant miRNA construct; (lane 3) RNAs from cells transfected with the miRNA expression vector fU1-miR. U2 and U6 snoRNA was used as an RNA loading control. Quantification of the signal was first normalized to U2, and then it was further normalized to the band with the lowest signal.

FIGURE 2.

Functional test of miR-510, miR-510-T/C, and miR-510-G/A. (A) Sequences of miRNAs and reporters. Stem–loop sequences of miR-510, miR-510-T/C, and the sequences inserted into the 3′UTR of Rluc for the “si” and “mi” reporters. (B) Transfection test results of T/C mutant. The knockdown of reporters for 5p-si, 5p-mi, 3p-si, and 3p-m-si (mutant form) from the mutant form are reduced. The repression for 3p-mi is approximately the same for both the WT and the mutant. Each bar represents the average of at least three independent transfections with duplicate determinations for each construct. Error bars represent the SD. (C) Sequences of miRNAs and reporters. Stem–loop sequences of miR-510, miR-510-G/A, and the inserted sequences into the 3′UTR of Rluc for the “si” and “mi” reporters. (D) Transfection test results of miR-510-G/A. The knockdown of reporters for 5p-si, 5p-mi, 3p-si, and 3p-m-si (G/A mutant form) from the mutant are all increased. Each bar represents the average of at least three independent transfections with duplicate determinations for each construct. Error bars represent the SD. (E) Northern blot results. Blot was probed with the miR-510-5p probe (right) and miR-510-3p probe (left); U2 and U6 snoRNAs were used as RNA sample loading controls. (Lane 1) Transfected with fU1-miR; (lane 2) transfected with miR-510 WT; (lane 3) transfected with the miR-510-T/C mutant; (lane 4) transfected with fU1-miR-510-G/A. Quantification of the signal was first normalized to U2, and then it was further normalized to the band with the lowest signal.

FIGURE 3.

Functional test of miR-890 and miR-890-G/C. (A) Sequences of miRNAs and reporters. Stem–loop sequences of miR-890, miR-890-G/C, and the sequences inserted into the 3′UTR of Rluc for the “si” and “mi” reporters. (B) Transfection test results. The knockdown of reporters for 5p-si, 5p-mi, 3p-si, and 3pGC-si (mutant form) from the mutant form are reduced. The expression of the 3p-mi reporter is approximately the same for both the WT and mutant. Each bar represents the average of at least three independent transfections with duplicate determinations for each construct. Error bars represent the SD. (C) Northern blot results. Blot was probed with miR-890-5p, 3pGC, and 3p for samples transfected with fU1-miR (lane 1), fU1-miR-890 (lane 2), and fU1-miR-890-G/C (lane 3). U2 and U6 snoRNAs were used as RNA loading controls.

FIGURE 4.

Functional test of miR-892b and miR-892b-T/C. (A) Sequences of miRNAs and reporters. Stem–loop sequences of miR-892b, miR-892b-T/C, and the sequences inserted into the 3′UTR of Rluc for the “si” and “mi” reporters. (B) Transfection test results. The knockdown of reporters for 5p-si, 3p-si, 3pm-si (mutant form), and 3p-mi from the mutant form are reduced. Each bar represents the average of at least three independent transfections with duplicate determinations for each construct. Error bars represent the SD. (C) Northern blot results. Blot was hybridized with probes for miR-892b-5p, 3p, and 3pTC (mutant form) using samples transfected with fU1-miR (lane 1), fU1-miR-892b (lane 2), and fU1-miR-892b-T/C (lane 3). U2 and U6 snoRNAs were used as RNA loading controls.

FIGURE 5.

Functional test of miR-934 and miR-934-T/G. (A) Sequences of miRNAs and reporters. Stem–loop sequences of miR-934, miR-934-T/G, and the sequences inserted into the 3′UTR of Rluc for the “si” and “mi” reporters. (B) Transfection test results. The knockdown of 5p-si and 5p-mi reporters by the mutant were reduced and strong knockdown of the 3p-si and 3pm2-si reporters are observed from the mutant miRNA. Each bar represents the average of at least three independent transfections, with duplicate determinations for each construct. Error bars represent the SD. (C) Northern blot results. Blot was probed with a miR-934-5p probe (left) and 3p probe (right). U2 and U6 snoRNAs were probed as RNA gel loading controls. (Lanes 1,2) From cells transfected with fU1-miR; (lane 3) from cells transfected with fU1-miR-934; (lane 4) from cells transfected with the fU1-miR-934-T/G.