Targetome of U1-36 (“RHO_4_-1G>A_1”).

The endogenous U1 small nuclear RNA (U1-snRNA) plays a crucial role in splicing initiation through base-pairing to donor splice sites (5′-SSs). Likewise, modified U1s that carry a mutation-adapted 5′-terminal sequence have been demonstrated to rescue exon splicing when this is disrupted by genetic mutations within the 5′-SS. Given the base-pairing flexibility of the endogenous U1, the selectivity of modified U1s requires investigation. We developed a computational pipeline (Utargetome) that considers combinations of mismatches and alternative annealing registers to predict the transcriptome-wide binding sites (or targetome) of a U1. The pipeline accuracy was tested by recapitulating well-established alternative annealing registers and specificity for 5′-SSs in the predicted targetome of the human endogenous U1. It was then applied to analyse the targetome of 54 modified U1s that have been demonstrated to restore exon inclusion when affected by 5′-SS pathogenic mutations. While the targetome size was found to be wide-ranging, the off-target load appeared to be reduced for U1s targeting distal sites from the canonical U1-binding position. This feature was predicted also for a large set of 30,204 newly designed U1s targeting 839 5′-SS pathogenic mutations that were expected to affect exon inclusion. Targetome analysis indeed revealed an optimal distal-targeting position at 3 nucleotides downstream from the canonical 5′-SS, for which a modified U1 is likely to have minimal off-targets at 5′-SSs and acceptor splice sites (3′-SSs). Based on these insights, we propose to implement targetome prediction in the design and optimization of therapeutic U1s with improved selectivity.内源性U1小核RNA（U1-snRNA）通过与剪接供体位点（5′-SSs）碱基配对，在剪接起始过程中发挥关键作用。同样，携带突变适配型5′末端序列的改造型U1（modified U1s），已被证实可挽救因5′-SSs内部基因突变引发的外显子剪接缺陷。鉴于内源性U1-snRNA的碱基配对灵活性，改造型U1的结合选择性亟待研究。我们开发了一款名为Utargetome的计算管线（computational pipeline），该管线可综合考量错配与可变退火配对位点的组合情况，进而预测U1的全转录组结合位点（即靶标组，targetome）。通过在人类内源性U1-snRNA的预测靶标组中重现已被广泛证实的可变退火配对位点以及对5′-SSs的结合特异性，验证了该计算管线的准确性。随后，我们将该管线应用于分析54种改造型U1的靶标组，这类改造型U1已被证实可恢复受5′-SSs致病性突变影响的外显子保留现象。研究发现，靶标组的规模跨度较大，但对于靶向经典U1结合位点远端区域的U1而言，其脱靶负载显著降低。这一特征在针对30204种全新设计的改造型U1的大规模预测中同样得到了验证，这些U1靶向839个预期会影响外显子保留的5′-SSs致病性突变。靶标组分析进一步揭示，在经典5′-SS下游3个核苷酸处存在最优的远端靶向位点，携带此类位点的改造型U1在5′-SSs与剪接受体位点（3′-SSs）处的脱靶效应极有可能降至最低。基于上述研究发现，我们提出在治疗性U1的设计与优化过程中引入靶标组预测方法，以提升其结合选择性。