Figure 2

APOBEC3G likely functions through DND1 to restore miRNA activity. (a) APOBEC3G blocks DND1 to restore miR-221 inhibition of luciferase from wild-type P27-3’-UTR (lane 4). (*) miR-221 inhibited luc-P27 expression (P = 0.0116) (lane 2), (#) DND1 rescued the inhibition (P= 0.0138) (lane 3). ($) APOBEC3G opposed the function of DND (P = 0.0467) (lane 4). (b) APOBEC3G does not restore miR-221 inhibition from mutated P27 3’-UTR in m1 (or luc-m1; in which both DND1 binding sites mutated) [4] (lane 4). (*) miR-221 inhibited luc-m1 expression (P= 0.0460). DND1 does not rescue miR-221inhibition (P=0.2665) (lane 3) and A3 does not affect DND1 function (P = 0.3211). (c) miR-221 does not inhibit m3 (or luc-m3, both miRNA binding sites mutated [4]) (P= 0.2665) (lane 2). DND1 (P= 0.1184) (lane 3) or A3 (P= 0.4569) (lane 4) also do not affect luc-m3 activity. (d) Effect of increasing levels of APOBEC3G on wild-type pGL3-P27-3’-UTR. APOBEC3G was cotransfected at two concentrations (10 ng and 25 ng). Higher APOBEC3G (A3) (25 ng) inhibits (*) pGL3-P27-3’-UTR (lane 3) (P=0.023). (e) Higher A3 (25 ng) slightly enhances luc-m1 expression (*) (lane 3) (both DND1 binding sites mutated) (P=0.0432). (f) Higher A3 (25 ng) also enhances luc-m3 expression (*) (lane 3) (both miR-221 binding sites mutated) (P = 0.0141). However, lower A3 levels (10 ng) have no effect on pGL3-P27-3’-UTR, luc-m1, luc-m3 expression (lane 2 in d, e and f: P= 0.0798, 0.3567 and 0.4544 respectively). (g) Effect of increasing APOBEC3G on miR function. miR-221 inhibits luc-P27 (P = 0.0153) (lane 2). A3 (10 ng), together with miR-221, does not further inhibit luc-P27 (P = 0.4553). However, A3 at 25 ng (lane 4), together with miR-221, further inhibits luc-P27 (P= 0.0488). (h) miR-206 inhibits luc-CX43 (P=0.0105) (lane 2). A3 (10 ng), together with miR-221 does not further inhibit luc-CX43 (P = 0.4765). However, 25 ng A3 (*) (lane 4), together with miR-221, further inhibits luc-CX43 (P= 0.0385). (i) miR-372 inhibits luc-LATS2 (P= 0.0011) (lane 2). A3 (10 ng), together with miR-221 does not further inhibit luc-LATS2 (P= 0.1673). However, 25 ng A3 (*) (lane 4), together with miR-221, further inhibits luc-LATS2 (P = 0.013). (j) Increasing levels of APOBEC3G have no effect on luciferase translation. Control luciferase reporter, pGL3-luciferase (1 ng) was cotranfected with increasing concentrations (10 ng and 25 ng) of APOBEC3G expression constructs (P= 0.4021 and P = 0.4573 respectively).