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. Overall design: 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).