RiboMeth-Seq analysis of purified U6 and U2 snRNAs

The La-related protein LARP7 has been mainly described as a component of the 7SK small nuclear ribonucleoprotein (snRNP) complex, which negatively regulates RNA polymerase II by sequestering the positive transcription elongation factor b (P-TEFb). In our studies, we discovered a novel, 7SK snRNP-independent function of LARP7. We show that LARP7 interacts with the U6 spliceosomal RNA as well as with the small nucleolar RNAs (snoRNAs) directing the 2'-O-methylations of U6. To investigate the relevance of this interaction, U6 or U2 snRNAs were purified from total RNA by pulldown of biotinylated antisense oligonucleotides and the occurence of 2’-O-methylations was investigated by RiboMeth-seq analysis. A comparison between U6 and U2 snRNA isolated from HEK293 wildtype or LARP7 knockout cell lines revealed that 2’-O-methylations of the U6 snRNA are specifically lost in the absence of LARP7. Alazami syndrome is a form of primary dwarfism associated with mutations in the LARP7 gene. RiboMeth-seq analyses performed with RNA isolated from blood samples of two Alazami patients or healthy parents as well as from B-lymphoblastoid cell lines (B-LCLs) derived from an Alazami patient and from a healthy parent confirmed the impact of mutant LARP7 protein variants on the 2’-O-methylation profile of the U6 snRNA. Three biological replicates were performed with the U6 snRNA purified from wildtype HEK293 cell and two biological replicates each from two independent HEK293 LARP7 knockout clones generated with the CRISPR/Cas9 system. 2’-O-methylations of the U2 snRNAs were analyzed from one sample derived from wildtype HEK293 cells and from two independent HEK293 LARP7 knockout clones (one sample each). HEK293 T-Rex LARP7 ko cell lines stably overexpressing FLAG/HA-tagged LARP7 wildtype or F44A mutants were assayed in two biological replicates each for U6 2’-O-methylations. U6 2’-O-methylations were also analyzed from material purified from human blood samples. Therefore, U6-snRNA originating from two siblings carrying homozygous loss-of-function mutations in the LARP7 gene and from both heterozygous parents was used for RiboMeth-Seq experiments. RiboMeth-Seq analyses of purified U6 snRNAs were also performed from total RNA extracted from two B-LCL cell lines originating either from a boy affected by the Alazami syndrome or from his non-affected father (two replicates each).

La相关蛋白LARP7（La-related protein LARP7）既往被广泛认为是7SK小核核糖核蛋白（small nuclear ribonucleoprotein，snRNP）复合物的组成成分，该复合物可通过螯合正向转录延伸因子b（positive transcription elongation factor b，P-TEFb）对RNA聚合酶II产生负调控作用。在本研究中，我们发现了LARP7的一种新型、不依赖7SK snRNP的功能。我们证实，LARP7可与U6剪接体RNA以及介导U6 2'-O-甲基化修饰的小核仁RNA（small nucleolar RNAs，snoRNAs）相互作用。
为探究该相互作用的生物学相关性，我们通过生物素标记反义寡核苷酸下拉实验从总RNA中纯化得到U6或U2小核RNA，并通过RiboMeth测序（RiboMeth-seq）分析检测2'-O-甲基化修饰的存在情况。对比从HEK293野生型或LARP7基因敲除细胞系中分离得到的U6与U2小核RNA，结果显示，U6小核RNA的2'-O-甲基化修饰在LARP7缺失时发生特异性丢失。
阿拉扎米综合征（Alazami syndrome）是一类与LARP7基因突变相关的原发性侏儒症。我们对两名阿拉扎米综合征患者及其健康父母的血液样本、以及从一名阿拉扎米综合征患者和一名健康亲本构建的B淋巴母细胞系（B-lymphoblastoid cell lines，B-LCLs）中提取的RNA开展了RiboMeth-seq分析，证实了突变型LARP7蛋白变体对U6小核RNA的2'-O-甲基化谱具有显著影响。
本研究针对从野生型HEK293细胞纯化得到的U6小核RNA设置了3次生物学重复；针对利用CRISPR/Cas9系统构建的两株独立的HEK293 LARP7基因敲除克隆，分别设置2次生物学重复。我们对1份野生型HEK293细胞样本以及两株独立的HEK293 LARP7基因敲除克隆（每株克隆各1份样本）中提取的U2小核RNA的2'-O-甲基化修饰进行了分析。针对稳定过表达FLAG/HA标签融合的野生型LARP7或F44A突变体的HEK293 T-Rex LARP7敲除细胞系，我们分别设置2次生物学重复，检测其U6小核RNA的2'-O-甲基化修饰水平。
我们还从人类血液样本纯化得到的材料中分析了U6小核RNA的2'-O-甲基化修饰：本次实验使用了携带LARP7基因纯合功能丧失性突变的两名同胞及其两名杂合子父母的U6小核RNA进行RiboMeth-seq实验。此外，我们从两株B-LCL细胞系（分别来自一名罹患阿拉扎米综合征的男孩及其未患病的父亲）提取的总RNA中纯化得到U6小核RNA并开展RiboMeth-seq分析，每株细胞系各设置2次生物学重复。