Fig 4. Expression of microRNA-548ac in vitro and effect of SNP rs1414273.

Precursor miRNA expression plasmids were used to express hsa-miR-548ac in HeLa cells and to examine allele-specific differences. (A) Sanger DNA sequencing chromatograms for the sense strand of the original plasmid (mir-548ac-G) and the mutated plasmid (mir-548ac-A), which only differ at a single base 11 nt downstream of the 3' Drosha cleavage site. The two plasmids represent the SNP rs1414273 at the lower stem of the primary miRNA foldback structure. The G allele (in blue) is the MS-associated allele. The sequence of the mature miRNA is underlined. (B) Real-time PCR measurement of hsa-miR-548ac molecules that were produced by the endogenous miRNA biogenesis machinery in HeLa cells. The levels of the mature miRNA were roughly twice as high after 48 h than after 24 h post-transfection with either plasmid. (C) Relative expression of hsa-mir-548ac precursor transcripts after transient transfection. A more than 10-fold decrease in the precursor levels was consistently observed from the 24 h to the 48 h time point. (D) Functional analysis of the possible effect of the genetic variant on hsa-mir-548ac processing efficiency in HeLa cells. At the 48 h time point, a non-significant 3.4-fold higher ratio of mature miRNA levels versus precursor RNA levels was found in mir-548ac-G-transfected cells compared with mir-548ac-A-transfected cells. The PCR data suggest that the A allele might be associated with reduced stem-loop recognition and/or processing by the Drosha-DGCR8 complex. Each bar plot displays the arithmetic means and standard errors of the scaled 2^-ΔCt (B and C) or 2^-ΔΔCt (D) values. MS = multiple sclerosis, PCR = polymerase chain reaction, SNP = single-nucleotide polymorphism.