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| Status |
Public on Feb 03, 2020 |
| Title |
RiboMeth-Seq analysis of purified U6 and U2 snRNAs |
| Organism |
Homo sapiens |
| Experiment type |
Other
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| Summary |
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.
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| 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).
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| Contributor(s) |
Hasler D, Lehmann G, Eichner N, Kunstmann E, Meister G |
| Citation(s) |
32017898 |
| |
| Submission date |
Feb 14, 2019 |
| Last update date |
Feb 19, 2020 |
| Contact name |
Gerhard Lehmann |
| E-mail(s) |
gerhard.lehmann@vkl.uni-regensburg.de
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| Organization name |
University of Regensburg
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| Department |
Biochemistry 1
|
| Street address |
Universitaetsstrasse 31
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| City |
Regensburg |
| ZIP/Postal code |
93053 |
| Country |
Germany |
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| Platforms (2) |
| GPL15433 |
Illumina HiSeq 1000 (Homo sapiens) |
| GPL15520 |
Illumina MiSeq (Homo sapiens) |
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| Samples (12)
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| GSM3608778 |
HEK293 U6 snRNA RiboMeth-Seq replicates 1-3 |
| GSM3608779 |
HEK293 LARP7 ko U6 snRNA RiboMeth-Seq replicates 1-4 |
| GSM3608780 |
HEK293 U2 snRNA RiboMeth-Seq |
| GSM3608781 |
HEK293 LARP7 ko U2 snRNA RiboMeth-Seq replicates 1-2 |
| GSM4216445 |
HEK293 T-Rex LARP7 ko stable FH-LARP7 WT rescue U6 snRNA RiboMeth-Seq replicates 1-2 |
| GSM4216446 |
HEK293 T-Rex LARP7 ko stable FH-LARP7 F44A rescue U6 snRNA RiboMeth-Seq replicates 1-2 |
| GSM4216447 |
U6 snRNA RiboMeth-Seq from human blood of an healthy parent 1 |
| GSM4216448 |
U6 snRNA RiboMeth-Seq from human blood of an healthy parent 2 |
| GSM4216449 |
U6 snRNA RiboMeth-Seq from human blood of a sibling affected by Alazami syndrome 1 |
| GSM4216450 |
U6 snRNA RiboMeth-Seq from human blood of a sibling affected by Alazami syndrome 2 |
| GSM4216451 |
U6 snRNA RiboMeth-Seq from B-LCL cell line originating from a healthy parent |
| GSM4216452 |
U6 snRNA RiboMeth-Seq from B-LCL cell line originating from a boy affected by the Alazami syndrome |
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| This SubSeries is part of SuperSeries: |
| GSE126911 |
The Alazami Syndrome-associated protein LARP7 guides U6 small nuclear RNA modification and contributes to splicing robustness |
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| Relations |
| BioProject |
PRJNA522469 |
| SRA |
SRP185940 |
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