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. 2020 Jun 2;48(13):7135–7153. doi: 10.1093/nar/gkaa460

Figure 3.

Figure 3.

DICER binds and processes major satellite repeat transcripts. (A) DICER interacts with MSR transcripts (MSR-tr) in vitro. DIG-labelled in vitro transcribed MSR or randomly-selected control transcripts were incubated with DICER-complexes (DCR) immunoprecipitated from mouse testes. Rabbit IgG (IgG) was used as a negative control for immunoprecipitation. Binding was detected by anti-DIG antibody both by dot blotting (left panel) or by running bound RNA into an agarose gel followed by transfer to a membrane (right panel). Both MSR-tr and control-tr bound to DICER complexes, while no binding was detected in the negative IgG control. (B) DICER complexes are able to process MSR transcripts in vitro. DIG-labeled MSR or control transcripts were incubated with testis DICER-complexes and RNA was run into a polyacrylamide gel for anti-DIG detection. Both MSR and control transcripts were processed into smaller products by DICER complexes (black arrow) but not by control IgG immunoprecipitations. (C) MSR transcripts form complexes with DICER in the testis in vivo. Cross-linked testicular extracts were immunoprecipitated by anti-DICER or control rabbit IgG antibodies, and co-precipitated RNAs were detected by RT-PCR using primers specific for MSR transcripts. L19 and Ppia (peptidylprolyl isomerase A) primers were used as negative controls. In the -RT reaction, cDNA synthesis reaction was performed in the absence of reverse transcriptase. (D) Long MSR transcripts accumulate in Dicer1 cKO testes. Total RNA was extracted from control (CTRL) and Dicer1 cKO (cKO) testes and run into an agarose gel for Northern blotting using DIG-labeled MSR forward transcript probes. The longer MSR transcripts that appear only in cKO testes are indicated by an asterisk. Total RNA was stained to visualize 18S and 5S rRNAs for the validation of equal loading of RNA. (E) MSR transcript-derived small RNAs are dramatically reduced in Dicer1 cKO testes. Small RNAs from control and Dicer1 cKO testes were run into a polyacrylamide gel and MSR forward transcript-derived small RNAs (asterisk) were detected by Northern blotting. Small RNAs were stained to visualize ∼30 nt piRNAs (black arrow) for the validation of equal loading of RNA. All panels show a representative figure of an experiment that was independently repeated at least two times.