Dicer partner protein tunes the length of miRNAs using base-mismatch in the pre-miRNA stem

Abstract

Dicer partner proteins Drosophila Loquacious-PB (Loqs-PB) and human TRBP tune the length of miRNAs produced by Dicer from a subset of pre-miRNAs and thereby alter their target repertoire, by an unknown mechanism. Here, we developed a novel high-throughput method that we named Dram-seq (Dice randomized pre-miRNA pool and seq) to study length distributions of miRNAs produced from thousands of different pre-miRNA variants. Using Dram-seq, we found that a base-mismatch in the pre-miRNA stem can alter the length of miRNAs compared with a base-pair at the same position in both Drosophila and human, and is important for the miRNA length tuning by Loqs-PB. Loqs-PB directly bound base-mismatched nucleotides in the pre-miRNA stem. We speculate that Loqs-PB tunes miRNA length by changing the conformation of base-mismatched nucleotides in the pre-miRNA stem to that of base-paired ones and thereby altering the distance of the pre-miRNA stem.

Figure 1.

Loqs-PB, but not Loqs-PA, binds the stem of pre-miR-307a and tunes the length of miR-307a. (A) Domain structures of Drosophila Loqs-PA, Drosophila Loqs-PB, human PACT, and human TRBP. Drosophila Loqs-PB has additional 46 aa residues in the linker between the second and third dsRBDs, compared with Loqs-PA. (B) In vitro dicing of 100 nM pre-miR-307a by 8 nM DmDicer-1 ± Loqs-PA or Loqs-PB for 120 min. 5′ arm-derived products (miR-307a-5p isomiRs), 3′ arm-derived products (miR-307a-3p isomiRs), and the terminal loop-derived products were detected by northern blot. DNA oligo probes 5′-CACACCCAGGTTGAGTGAGT-3′, 5′-TCGCTCACTCAAGGAGGTTG-3′, and 5′-TGGATACCATATCGAAATAA-3′ were used to detect miR-307–5p, miR-307–3p, and terminal loop, respectively. Representative gel images and length distributions revealed by quantification of gels (means of at least three independent trials) are shown. (C) In vitro dicing of 100 nM dsRNAs by 8 nM DmDicer-1 ± Loqs-PA or Loqs-PB for 120 min. The top strands are 5′ ³²P-radiolabeled. The other end of the top strands was blocked by two deoxynucleotides (30). Representative gel images and length distributions revealed by quantification of gels (means of at least three independent trials) are shown. (D) Site-specific UV crosslinking of 5′ ³²P-radiolabeled pre-miR-307a to 10 nM DmDicer-1 + Loqs-PA or Loqs-PB. Pre-miR-307a containing 5IU nucleotide modification at nucleotide positions 13, 52, 33 and 65 were used from left to right.

Figure 2.

Dram-seq. (A) Schematic representation of Dram-seq. An example using the 5′ arm-randomized pre-miRNA variants pool is shown. (B) Length distributions of miR-307a-5p isoforms produced from wild-type pre-miR-307a and its variants containing a single point mutation at nucleotide positions 1–19 by DmDicer-1 ± Loqs-PA or Loqs-PB, revealed by Dram-seq.

Figure 3.

miR-307a length distributions revealed by Dram-seq, individual gel-based in vitro assay, and sequencing from fly ovaries. (A) In vitro dicing of 100 nM pre-miR-307a variants by 8 nM DmDicer-1 ± Loqs-PA or Loqs-PB for 120 min. The miR-307a isoforms were detected by northern blot (top rows); length distributions revealed by quantification of gels (means of at least three independent trials) (middle rows); length distributions revealed by Dram-seq (bottom rows). (B) Length distributions of miR-307a isoforms produced from wild-type pre-miR-307a and its variants by recombinant DmDicer-1 ± Loqs-PA or Loqs-PB in vitro revealed by Dram-seq (left threes). Length distributions of miR-307a isoforms produced from the same pre-miR-307 variants in fly ovaries (right). Fly strains w¹¹¹⁸; miR-307a^null; UASP-miR-307a transgenes [wild-type and point mutants]/mat-15-Gal4.

Figure 4.

Mean length of miR-307 isoforms produced from pre-miR-307a variants revealed by Dram-seq. (A, B) Mean of mean length of miRNAs produced by DmDicer-1 ± Loqs-PA or Loqs-PB from the (A) 5′ arm- and (B) 3′ arm- randomized pre-miR-307a variants containing a base-pair or base-mismatch at each position in the stem. The pre-miR-307a variants containing up to one mutation in addition to the mutation in the position of interest were analyzed. Orange and green stars at stem positions 11 and 13 indicate that the mean length was shorter and longer, respectively, when the positions were base-paired, compared with base-mismatched. (C, D) Mean of the difference of mean miRNA length produced from the (C) 5′ arm- and (D) 3′ arm- randomized pre-miR-307a variants containing a base-pair or base-mismatch at each position in the stem between DmDicer-1 + Loqs-PA and DmDicer-1 alone or between DmDicer-1 + Loqs-PB and DmDicer-1 alone. The difference in mean miRNA length was examined for each unique pre-miR-307a variant, and their means are shown. The stem position numbers are shown in parentheses in (B) and (D). Orange and green stars at stem positions 11 and 13 indicate that the Loqs-PB effect to make longer miRNAs was reduced when these positions were base-paired, compared with base-mismatched.

Figure 5.

Dram-seq using human Dicer, PACT, TRBP, and pre-miR-132. (A) In vitro dicing of 100 nM 5′ ³²P-radiolabeled pre-miR-132, pre-miR-27b, and their swap chimera pre-miRNAs by 6.5 nM HsDicer ± PACT or TRBP for 120 min. Representative gel image and length distributions revealed by quantification of gels (means of at least three independent trials) are shown. (B) Length distributions of miR-132a-3p isoforms produced from wild-type pre-miR-132 and its variants containing a single point mutation at nucleotide positions 40–58 by HsDicer ± PACT or TRBP, revealed by Dram-seq.

Figure 6.

Mean length of miR-132 isoforms produced from pre-miR-132 variants revealed by Dram-seq. (A, B) Mean of mean length of miRNAs produced by HsDicer ± PACT or TRBP from (A) 5′ arm- and (B) 3′ arm- randomized pre-miR-132 variants containing a base-pair or base-mismatch at each position in the stem. The pre-miR-132 variants containing up to one mutation in addition to the mutation in the position of interest were analyzed. Green stars at the stem position 13 indicate that mean length was shorter, when the position was base-mismatched, compared with base-paired. (C, D) Mean of the difference of mean miRNA length produced from the (C) 5′ arm- and (D) 3′ arm- randomized pre-miR-132 variants containing a base-pair or base-mismatch at each position in the stem between HsDicer + PACT and HsDicer alone or between HsDicer + TRBP and HsDicer alone. The difference in mean miRNA length was examined for each unique pre-miR-132 variant, and their means are shown. Orange and green stars at stem positions 11 and 13 indicate that the Loqs-PB effect to make longer miRNAs was reduced when these positions were base-paired, compared with base-mismatched. The stem position numbers are shown in parentheses in (B) and (D).

Figure 7.

Conversion of a Loqs-PB-non-responsive pre-miRNA into Loqs-PB-responsive by introducing base-mismatches in the pre-miRNA stem. (A) Drosophila pre-miR-999. Nucleotides C45 and C53, which were mutated in (D), are shown in red. (B) Mean of mean miRNA length produced by DmDicer-1 ± Loqs-PA or Loqs-PB from 3′ arm-randomized pre-miR-999 variants containing a base-pair or base-mismatch at each position in the stem determine by Dram-seq. The pre-miR-999 variants containing up to one mutation in addition to the mutation in the position of interest were analyzed. Green stars at stem positions 5, 6 and 14 indicate that the mean length was longer, when these positions were base-mismatched, compared with base-paired. (C) Mean of the difference of mean miRNA length produced from pre-miR-999 variants containing a base-pair or base-mismatch at each position in the stem between DmDicer-1 + Loqs-PA and DmDicer-1 alone or between DmDicer-1 + Loqs-PB and DmDicer-1 alone. The difference in mean miRNA length was examined for each unique pre-miR-999 variant, and their means are shown. Green stars at stem positions 5, 6, and 14 indicate that Loqs-PB promoted production of shorter miRNA isoforms than DmDicer-1 ± Loqs-PA when these positions were base-mismatched. The stem position numbers are shown in parentheses in (B) and (C). (D) Length distributions of miRNA isoforms produced from the wild-type and double point mutants of pre-miR-999 determined by Dram-seq. (E) The speculative model by which Loqs-PB tunes the length of miRNAs using base-mismatches in the pre-miRNA stem. The base-mismatched nucleotides at stem position 13 in pre-miR-307a take extended conformation and thus make the pre-miR-307a stem length longer compared with base-paired (Middle). Therefore, fewer pre-miR-307a stem nucleotides can fit in the Dicer molecular ruler, resulting in production of shorter miR-307a isoform (21 nt) by DmDicer-1 ± Loqs-PA compared with pre-miRNA variants containing a base-pair at the stem position 13 (Top). Loqs-PB binding changes the conformation of the base-mismatched nucleotides at stem position 13 of pre-miR-307a to that of base-paired ones (Middle). Therefore, upon binding of Loqs-PB, the length of the pre-miR-307a stem becomes shorter as in base-paired pre-miRNAa, resulting in production of longer miR-307a isoforms (22 and 23 nt) compared with DmDicer-1 ± Loqs-PA. In a certain context, base-mismatched nucleotides can take a more compact conformation, shorten the pre-miRNA stem, and results in production of longer miRNA isoforms. Upon Loqs-PB binding, their conformation is changed to that of base-paired ones, and the pre-miRNA stem becomes shorter, resulting in production of shorter miRNA isoforms compared with DmDicer-1 ± Loqs-PA. Most of mismatches in the pre-miRNA stem do not change the pre-miRNA stem length or miRNA length.