Figure 2

Experimental validation of miR-H3. (A) Mature miRNA sequences revealed by deep sequencing. Primary human CD4⁺ T-lymphocytes isolated from healthy donors were activated with anti-CD3(1 μg/ml) and anti-CD28(5 μg/ml) antibodies and infected with wildtype HIV-1_NL4-3 viruses. The total RNAs from infected or uninfected CD4⁺ T-lymphocytes were extracted for deep sequencing on small RNA profiling. (B) RNase protection assay of small RNAs (<200 nt) isolated from primary CD4⁺ T-lymphocytes infected with wild type HIV-1_NL4-3 viruses or uninfected CD4⁺ T-lymphocytes. The probe is corresponding to 34-71 nt of the miR-H3 precursor. U6 RNA was detected as the loading control for RPA. (C) Real-time qRT-PCR was conducted on total RNAs isolated from infected or uninfected activated CD4⁺ T-lymphocytes with primers for miR-H3-3p and miR-H3-5p respectively. MiR-31 was set as a positive control, while U6 RNA expression was set as the internal control and set as 1 unit. (D) Two key factors in miRNA processing, DROSHA and XPO5, were knocked-down with siRNAs, and the expression of miR-H3-3p from its precursor transfected into HEK293T was investigated by qRT-PCR as described above. (E) Subcellular localization of miR-H3. HEK293T cells were transfected with pCMV-ΔR8.2 vector. Fouty-eight hrs later, nuclear and cytoplasmic RNAs were isolated separately and the expression of tRNA-Gly, snoRNA-snoRD57 and miR-H3-3p were tested with real-time qRT-PCR. P-values were calculated using the two tailed unpaired Student’s t-test with equal variances, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.