Figure 1. pre-miR-128-2 expression precedes miR-128.
Northern blots of RNA from embryonic and adult mouse brains. RNA from the stages indicated above each lane was hybridized with probes specific for miR-128 (A); pre-miR-128-2 (B); and U6 (C) as loading control. The position of precursor RNAs is indicated with a filled arrow, the ∼21 nt miRNA with an open arrow. The portion of the filter corresponding to ∼15 to 100 nt is shown. The pre-miR-128-2 sequence is depicted in (D), showing the 21 nt mature sequence that is targeted by the anti-miR-128 LNA probe (underlined) and the sequence complementary to the anti-precursor hybridization probe (red).
Figure 1—figure supplement 1. Relative activity of pre-miR-128-1-RED and pre-miR-128-2-RED expression constructs.
(A) The sequences of pre-miR-128-1 (top) and pre-miR-128-2 (bottom) are depicted, showing the mature 21 nt miRNA sequence as recognized by the anti-miR-128 LNA probe (underlined) and the sequence complementary to the anti-precursor hybridization probe (red). (B) Northern blots of RNA from HEK-293 cells transfected with Intron-RED empty vector (Lane 1), pre-miR-128-1-RED (Lane 2) or pre-miR-128-2-RED (Lane 3). The filter was hybridized with probes specific for mature miR-128, pre-miR-128-1, pre-miR-128-2 or U6 RNA as loading control, as indicated above each panel. The position of precursor RNAs is indicated with a filled arrow, the 21 nt miR-128 with an open arrow. The portion of the filter corresponding to approximately 15–100 nt is shown. The pre-miR-128-1-RED expression vector produces less miR-128 than the pre-miR-128-2-RED vector, each probe shows the expected specificity. (C) HEK-293 cells were co-transfected with a GFP-based sensor vector containing four perfectly complementary binding sites for miR-128 and either Intron-RED control vector, pre-miR-128-1-RED or pre-miR-128-2-RED expression vectors, as indicated. Both pre-miR-128-RED expression constructs repress the miR-128 sensor but pre-miR-128-1-RED shows less activity than pre-miR-128-2-RED.
Figure 1—figure supplement 2. Levels of pre-miR-128-1 are below detection level in Northern blot and in situ hybridization assays.
(A) Northern blot as in Figure 1A–C, the membrane in this case was hybridized with the pre-miR-128-1 probe (described in Figure 1—figure supplement 1). Developmental stage of the RNA is indicated above each lane. (B–B″′) In situ hybridization using the pre-miR-128-1 probe (see Figure 1—figure supplement 1) of embryonic day 12.5 (B), 16.5 (B′), 18.5 (B″) and Adult (B″′) brains. The obtained signal does not exceed background at any time point examined.