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@article{1550500,
author = {Souček, Přemysl and Réblová, Kamila and Kramárek, Michal and Radová, Lenka and Grymová, Tereza and Hujová, Pavla and Kováčová, Tatiana and Lexa, Matej and Grodecká, Lucie and Freiberger, Tomáš},
article_location = {PHILADELPHIA},
article_number = {10},
doi = {http://dx.doi.org/10.1080/15476286.2019.1630796},
keywords = {SMN1; cryptic splice sites; U1 snRNA; splicing-affecting mutation; 5 ' ss},
language = {eng},
issn = {1547-6286},
journal = {RNA BIOLOGY},
title = {High-throughput analysis revealed mutations' diverging effects on SMN1 exon 7 splicing},
url = {http://dx.doi.org/10.1080/15476286.2019.1630796},
volume = {16},
year = {2019}
}
TY - JOUR
ID - 1550500
AU - Souček, Přemysl - Réblová, Kamila - Kramárek, Michal - Radová, Lenka - Grymová, Tereza - Hujová, Pavla - Kováčová, Tatiana - Lexa, Matej - Grodecká, Lucie - Freiberger, Tomáš
PY - 2019
TI - High-throughput analysis revealed mutations' diverging effects on SMN1 exon 7 splicing
JF - RNA BIOLOGY
VL - 16
IS - 10
SP - 1364-1376
EP - 1364-1376
PB - TAYLOR & FRANCIS INC
SN - 15476286
KW - SMN1
KW - cryptic splice sites
KW - U1 snRNA
KW - splicing-affecting mutation
KW - 5 ' ss
UR - http://dx.doi.org/10.1080/15476286.2019.1630796
L2 - http://dx.doi.org/10.1080/15476286.2019.1630796
N2 - Splicing-affecting mutations can disrupt gene function by altering the transcript assembly. To ascertain splicing dysregulation principles, we modified a minigene assay for the parallel high-throughput evaluation of different mutations by next-generation sequencing. In our model system, all exonic and six intronic positions of the SMN1 gene's exon 7 were mutated to all possible nucleotide variants, which amounted to 180 unique single-nucleotide mutants and 470 double mutants. The mutations resulted in a wide range of splicing aberrations. Exonic splicing-affecting mutations resulted either in substantial exon skipping, supposedly driven by predicted exonic splicing silencer or cryptic donor splice site (5 ' ss) and de novo 5 ' ss strengthening and use. On the other hand, a single disruption of exonic splicing enhancer was not sufficient to cause major exon skipping, suggesting these elements can be substituted during exon recognition. While disrupting the acceptor splice site led only to exon skipping, some 5 ' ss mutations potentiated the use of three different cryptic 5 ' ss. Generally, single mutations supporting cryptic 5 ' ss use displayed better pre-mRNA/U1 snRNA duplex stability and increased splicing regulatory element strength across the original 5 ' ss. Analyzing double mutants supported the predominating splicing regulatory elements' effect, but U1 snRNA binding could contribute to the global balance of splicing isoforms. Based on these findings, we suggest that creating a new splicing enhancer across the mutated 5 ' ss can be one of the main factors driving cryptic 5 ' ss use.
ER -
SOUČEK, Přemysl, Kamila RÉBLOVÁ, Michal KRAMÁREK, Lenka RADOVÁ, Tereza GRYMOVÁ, Pavla HUJOVÁ, Tatiana KOVÁČOVÁ, Matej LEXA, Lucie GRODECKÁ and Tomáš FREIBERGER. High-throughput analysis revealed mutations' diverging effects on SMN1 exon 7 splicing. \textit{RNA BIOLOGY}. PHILADELPHIA: TAYLOR \&{}amp; FRANCIS INC, vol.~16, No~10, p.~1364-1376. ISSN~1547-6286. doi:10.1080/15476286.2019.1630796. 2019.
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