RT Journal
A1 Erkelenz, Steffen
A1 Theiss, Stephan
A1 Kaisers, Wolfgang
A1 Ptok, Johannes
A1 Walotka, Lara
A1 Müller, Lisa
A1 Hillebrand, Frank
A1 Brillen, Anna-Lena
A1 Sladek, Michael
A1 Schaal, Heiner
T1 Ranking noncanonical 5′ splice site usage by genome-wide RNA-seq analysis and splicing reporter assays
JF Genome Research 
JO Genome Research 
YR 2018 
FD December 01 
VO 28 
IS 12 
SP 1826 
OP 1840 
DO 10.1101/gr.235861.118 
UL http://genome.cshlp.org/content/28/12/1826.abstract 
AB Most human pathogenic mutations in 5′ splice sites affect the canonical GT in positions +1 and +2, leading to noncanonical dinucleotides. On the other hand, noncanonical dinucleotides are observed under physiological conditions in ∼1% of all human 5′ss. It is therefore a challenging task to understand the pathogenic mutation mechanisms underlying the conditions under which noncanonical 5′ss are used. In this work, we systematically examined noncanonical 5′ splice site selection, both experimentally using splicing competition reporters and by analyzing a large RNA-seq data set of 54 fibroblast samples from 27 subjects containing a total of 2.4 billion gapped reads covering 269,375 exon junctions. From both approaches, we consistently derived a noncanonical 5′ss usage ranking GC > TT > AT > GA > GG > CT. In our competition splicing reporter assay, noncanonical splicing was strictly dependent on the presence of upstream or downstream splicing regulatory elements (SREs), and changes in SREs could be compensated by variation of U1 snRNA complementarity in the competing 5′ss. In particular, we could confirm splicing at different positions (i.e., −1, +1, +5) of a splice site for all noncanonical dinucleotides “weaker” than GC. In our comprehensive RNA-seq data set analysis, noncanonical 5′ss were preferentially detected in weakly used exon junctions of highly expressed genes. Among high-confidence splice sites, they were 10-fold overrepresented in clusters with a neighboring, more frequently used 5′ss. Conversely, these more frequently used neighbors contained only the dinucleotides GT, GC, and TT, in accordance with the above ranking.