Loss of the RNA trimethylguanosine cap is compatible with nuclear accumulation of spliceosomal snRNAs but not pre-mRNA splicing or snRNA processing during animal development. Loss of the RNA trimethylguanosine cap is compatible with nuclear accumulation of spliceosomal snRNAs but not pre-mRNA splicing or snRNA processing during animal development

The 2,2,7-trimethylguanosine (TMG) cap is synthesized by the conserved Tgs1 enzyme, which is abundantly present on snRNAs essential for pre-mRNA splicing. In our study, we characterize new mutations in the Drosophila tgs1 gene and studied their effects on development and on splicing. Utilizing a hypomorphic tgs1 mutation, we uncovered a prominent role of Tgs1 in pre-mRNA splicing, particularly of introns with smaller sizes and weaker splice sites. Remarkably, stronger tgs1 mutations generated by CRISPR-Cas9 cause lethality without disrupting splicing, likely due to the preponderance of a maternal contribution and stable TMG capped snRNAs maintaining a near normal level of TMG modified snRNAs, which further proved that the TMG modification is important in splicing. Overall design: 8 samples in total; 4 kinds of RNA samples from wildtype and tgs1 mutant animals were prepared and sequenced with two replicates.

2,2,7-三甲基鸟苷（2,2,7-trimethylguanosine，TMG）帽由保守的Tgs1酶催化合成，该酶在pre-mRNA剪接所必需的小核RNA（small nuclear RNA，snRNA）上大量富集。本研究对果蝇tgs1基因的新型突变进行了表征，并探究了其对个体发育及pre-mRNA剪接过程的影响。借助一株低功能型tgs1突变体，我们揭示了Tgs1在pre-mRNA剪接中的关键作用，尤其针对小型内含子与弱剪接位点的剪接事件。值得注意的是，通过CRISPR-Cas9技术构建的强效tgs1突变体可导致个体致死，但并未干扰剪接过程，这可能源于母体贡献的丰度及稳定的TMG帽修饰snRNA，使得经TMG修饰的snRNA维持在接近正常的水平，该结果进一步证实了TMG修饰在剪接过程中的重要性。总体实验设计：共包含8个样本；分别从野生型与tgs1突变体动物中制备4类RNA样本，并设置两个生物学重复进行测序。