FIGURE 4.

Influence of distal hairpin G:C content of 3′ resection efficiency. (A) mir-451 constructs reprogrammed with pre-mir-199a (mir-199a-RP) or pre-mir-375 (mir-375-RP). These were compared with chimeric constructs in which the hairpin stem distal to the Ago2 cleavage site (marked by the horizontal line) was exchanged. GC pairs in the resection region are marked with blue asterisks. (B,C) Northern analysis of the wild-type and chimeric constructs in HeLa cell or Dicer^−/− MEFs. Similar patterns were observed in these cell lines except that expression of the mir-451-type constructs is higher in the absence of Dicer, whereas expression of the linked mir-144 is arrested at the pre-miRNA stage. The blots show that the 3′ resection pattern is correlated with the identity of the hairpin region distal to the Ago2 cleavage site; i.e., 5′-375:199a-3′ resembles miR-199a, while 5′-199a:375-3′ resembles miR-375. (D) Direct manipulation of GC content in the human pre-mir-451 backbone. It normally contains two GCs in the 3′ resected region, and variant constructs sampled 3/4/5/8 GC pairs. (E) Northern analysis of the GC variant panel of mir-451. Increase of distal GC content impairs generation of 3′ resected forms of miR-451 in HeLa cells. The levels of Ago-cleaved and 3′ resected forms of miR-451 are severely reduced with high GC content. (*) The pre-mir-451, which appears to decrease with higher GC content. However, control experiments using oligonucleotide standards show that these high-GC-content hairpins are inefficiently detected; in contrast, the linear Ago-cleaved forms of these hairpins are detected with equal efficiency (Supplemental Fig. 4). Expression of miR-144 validates equal transfection and loading. (F) Functional consequences of increased GC content on miR-451 function. These were tested in Dicer^−/− cells against a luciferase sensor bearing four seed matches for miR-451. The variants with the highest GC content in the 3′ resected region were severely impaired in their repression activity.