Modeling splicing outcome by combining 5'ss strength and splicing regulatory elements.

Correct pre-mRNA processing in higher eukaryotes vastly depends on splice site recognition. Beyond conserved 5'ss and 3'ss motifs, splicing regulatory elements (SREs) play a pivotal role in this recognition process. Here, we present in silico designed sequences with arbitrary a priori prescribed splicing regulatory HEXplorer properties that can be concatenated to arbitrary length without changing their regulatory properties. We experimentally validated in silico predictions in a massively parallel splicing reporter assay on more than 3000 sequences and exemplarily identified some SRE binding proteins. Aiming at a unified 'functional splice site strength' encompassing both U1 snRNA complementarity and impact from neighboring SREs, we developed a novel RNA-seq based 5'ss usage landscape, mapping the competition of pairs of high confidence 5'ss and neighboring exonic GT sites along HBond and HEXplorer score coordinate axes on human fibroblast and endothelium transcriptome datasets. These RNA-seq data served as basis for a logistic 5'ss usage prediction model, which greatly improved discrimination between strong but unused exonic GT sites and annotated highly used 5'ss. Our 5'ss usage landscape offers a unified view on 5'ss and SRE neighborhood impact on splice site recognition, and may contribute to improved mutation assessment in human genetics.

Characterisation of Type II DNA Methyltransferases of Metamycoplasma hominis.

Bacterial virulence, persistence and defence are affected by epigenetic modifications, including DNA methylation. Solitary DNA methyltransferases modulate a variety of cellular processes and influence bacterial virulence; as part of a restriction-modification (RM) system, they act as a primitive immune system in methylating the own DNA, while unmethylated foreign DNA is restricted. We identified a large family of type II DNA methyltransferases in Metamycoplasma hominis, comprising six solitary methyltransferases and four RM systems. Motif-specific 5mC and 6mA methylations were identified with a tailored Tombo analysis on Nanopore reads. Selected motifs with methylation scores >0.5 fit with the gene presence of DAM1 and DAM2, DCM2, DCM3, and DCM6, but not for DCM1, whose activity was strain-dependent. The activity of DCM1 for CmCWGG and of both DAM1 and DAM2 for GmATC was proven in methylation-sensitive restriction and finally for recombinant rDCM1 and rDAM2 against a dam-, dcm-negative background. A hitherto unknown dcm8/dam3 gene fusion containing a (TA) repeat region of varying length was characterized within a single strain, suggesting the expression of DCM8/DAM3 phase variants. The combination of genetic, bioinformatics, and enzymatic approaches enabled the detection of a huge family of type II DNA MTases in M. hominis, whose involvement in virulence and defence can now be characterized in future work.