Fig. 1: Reevaluating the role of helicase in the loop counting rule.

a Schematic of two-loop shRNA featuring primary and secondary loops. A 32-nucleotide randomized sequence (32N) within the secondary loop functioned as a barcode. Green and red arrowheads mark DICER's double cleavage sites, DC21 and DC22, located 21 and 22 nucleotides from the shRNA's 5p-end, respectively. b Detection of shRNA variants in high-throughput dicing assays. Blue bars represent the count of shRNA variants identified per group. c Log₂-transformed distribution of barcode counts for shRNA variants found across two separate high-throughput dicing assays for DICERΔHelicase. d Consistency between two high-throughput dicing assays for DICERΔHelicase by calculating double cleavage efficiency for each shRNA variant. Each dot point corresponds to a single shRNA variant, with 'r' denoting Pearson's correlation coefficient. e Comparison of the accuracy between cleavages that are 2 nucleotides from a loop and those that are not in DICER-WT and DICERΔHelicase libraries. The upper panel depicts cleavage occurring 2 nucleotides away from the single-stranded region including terminal loop, internal loop, and bulges. This is designated as “2nt-loop” cleavage. Cleavage events that do not conform to this pattern are classified as “no-2nt-loop” to distinguish them from the “2nt-loop” cleavage process. DC20, DC21, and DC22 refer to double cleavages at the 20, 21, and 22 positions from the shRNA's 5p-end, respectively. Asterisks denote statistical significance (p < 0.05) obtained from two-tailed Mann-Whitney U test. The sample size was indicated in the boxplots. The median value (center line), lower quartile and upper quartile (box edges), and whiskers extended to ±1.5 × IQR (interquartile range) were indicated in each boxplot. f Calculation of DICER's double cleavage accuracy at positions 19–23 in shRNAs with the stem of 21–23 base pairs. Each graph line represents a randomized shRNA variant. g shRNA structures and sequences with green and red arrowheads indicating DC21 and DC22 cleavage sites, respectively. h In vitro DICER cleavage essays for RNAs depicted in (g). i Calculation of the difference in cleavage precision between DC22 and DC21 for DICER-WT and DICERΔHelicase from three repeated assays as shown in (h), the n.s indicate no statistical significant differences from the two-sided t-test. j sh-miR30 structures and sequences. k In vitro DICER cleavage essays for RNAs depicted in (j). l Assessment of cleavage precision at DC21 and DC22 for DICER-WT from three repeated assays as demonstrated in (k). m In vitro DICERΔHelicase cleavage essays for RNAs depicted in (j). n Assessment of cleavage precision at DC21 and DC22 for DICERΔHelicase from three repeated assays as demonstrated in (m). o A model illustrating the loop counting rule's independence from DICER's helicase domains. In the bar plot representing quantitative data from the gel, data values are shown as dots, and the error bars are presented with 95% confidence intervals.