MapToCleave: high-throughput profiling of microRNA biogenesis in living cells

We here introduce MapToCleave, a new method to simultaneously profile processing of thousands of distinct RNA structures in living cells. We find that miRNA precursors connected to a stable lower basal stem are more efficiently processed and also have higher expression in vivo in tissues from twenty animal species. We systematically compare the importance of known and novel sequence and structural features, and test miRNA precursors from ten animal and plant species in human cells. Last, we provide evidence that the known GHG motif better predicts processing when defined as a structure rather than sequence motif, consistent with recent cryo-EM studies. In summary, we apply a new screening assay in living cells to reveal the importance of lower basal stem stability for miRNA processing and in vivo expression. Two major experiments are included: 1) MapToCleave assay, where we applied in a single experiment to simultaneously profile the processing of 12,472 distinct RNA structures in living cells. These structures include bona fide human microRNA precursors, non-human microRNA precursors and control non-hairpin sequences. First, the test sequences were synthesized and cloned into an expression vector. The generated expression constructs were pooled in a single library and then transfected into human HEK-293T and HeLa cells and mouse NIH-3T3 and MEF cells. The successfully transfected individual vectors were identified by DNA sequencing. The test sequences were transiently expressed and the ones forming the correct hairpin structure were processed into small RNAs, which were captured by small RNA sequencing. Here we provide small RNA sequence data from HEK-293T mock cells, HEK-293T cells transfected with one time (1x) of the concentration of MapToCleave library, HEK-293T cells transfected with ten times (10x) of the concentration of MapToCleave library, HeLa mock cells, HeLa cells transfected with MapToCleave library, NIH-3T3 mock cells, NIH-3T3 cells transfected with 1x of MapToCleave library, MEF mock cells and MEF cells transfected with 1x of MapToCleave library. We provide DNA sequencing data from HEK-293T cells transfected with 1x of MapToCleave library and NIH-3T3 cells transfected with 1x of MapToCleave library. 2) Experimental validation of stability of lower basal stem tuning of microRNA processing, where we tested the processing of four variants of mir-16, one of which should stabilize the lower basal stem and improve processing (hsa-mir-16-1 variant 1) and three of which should destabilize the lower basal stem and impair processing (hsa-mir-16-1 variant 2-4). In each experiment we co-transfected with equimolar abundances of mir-30a and mir-125a for normalization, and all transfected miRNAs were additionally modified (tagged) in the mature region to discern them from endogenous miRNAs. We also tested the processing of three variants of mir-30a, two of which should stabilize the lower basal stem and improve processing (hsa-mir-30a variant 1-2) and one of which should destabilize the lower basal stem and impair processing (hsa-mir-30a variant 3). In each experiment we co-transfected with equimolar abundances of mir-16 and mir-125a for normalization, and all transfected miRNAs were additionally modified (tagged) in the mature region to discern them from endogenous miRNAs.