ILF3 is a positive regulator of pre-mir-144 dicing. (A–D) Knockdown assays in K562 cells (A-B) and HEK293T cells (C-D). (A) Independent lentiviral shRNA constructs were transduced into K562 cells. Target proteins were evaluated by Western blotting after 5 days of knockdown. shILF3-2 was slightly more effective than shILF3-1, while the two shBUD13 constructs were comparable. (B) Northern blotting shows that only shILF3 increased pre-mir-144, with a stronger effect in shILF3-2 cells (blue arrowhead) that exhibit stronger ILF3 depletion. (C) Quantification of pri-mir-144/451 transcripts following transfection in HEK293T cells. (D) Northern blotting of ectopic mir-144/451 expression shows attenuated pre-mir-144 dicing upon ILF3 depletion in HEK293T cells. (E) ENCODE K562 eCLIP data show that ILF3 and DGCR8 are selectively enriched at the mir-144 terminal loop (mir-22 shown for comparison). (F) ILF3 eCLIP data enriches a motif with sequence and structural similarity to the conserved motif in the pre-mir-144 loop. (G) RNA Bind-N-Seq (RBNS) analysis using ILF3 (NF90/NF110) proteins immunoprecipitated from HEK293T cells. Independent RBNS datasets enriched for similar sequences, which resemble the eCLIP motif and the conserved mir-144 loop. (H) ILF3 exhibits strong association to Dicer cofactor TRBP. Tagged constructs were co-expressed in HEK293T cells in the presence of mir-144/451 (WT144) or a version bearing mutations in the loop nucleotides (144LM). HA-NF90 was immunoprecipitated (IP-ed), and bound proteins were compared between input (In) and IP samples. NF90 is a known heterodimeric partner of NF45 (ILF2), and this association was robust. NF90 was also reported to bind DGCR8 and Exportin-5 (XPO5); we validated modest association to the former but not the latter. Amongst other miRNA factors, the Dicer cofactor TRBP was strongly co-IPed with NF90. Additional co-IP data are shown in Supplementary Figure S3C. (I) RNase treatments show that both NF90-NF45 and NF90-TRBP complexes are independent of RNA.
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