Project Details
Projekt
Deciphering the functions of human exon junction complexes
Subject Area
Cell Biology
Bioinformatics and Theoretical Biology
Bioinformatics and Theoretical Biology
Term
from 2019 to 2021
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 418083979
Splicing is not only an essential step for the maturation of most mammalian pre-mRNAs but also deposits exon junction complexes (EJC) on mRNAs. The EJC regulates several post transcriptional steps of gene expression, including mRNA localization, translation and turnover. Moreover, cells lacking EJC components display remarkable changes in splicing caused by unknown mechanisms. We have recently discovered that EJCs efficiently suppress splicing of nearby exonic splice sites (SS). Hereby, the EJC recruits the splicing regulator RNPS1 to inhibit cryptic 5' SS usage and to prevent exon skipping by re-splicing (also known as recursive splicing). In addition, the deposition of the EJC core directly masks reconstituted 3' SS and thereby precludes transcript disintegration. Thus, EJCs protect the transcriptome of mammalian cells from inadvertent loss of exonic sequences and thereby safeguard the expression of intact, full length mRNAs.During our experimental analysis we discovered that EJC-dependent splicing changes involve at least three additional factors - RNPS1, SAP18 and PNN. However, high-quality RNA-Seq data exist for RNPS1, but not for SAP18, PNN or the EJC core factors (EIF4A3, RBM8A, MAGOHA&B). Therefore, we plan to carry out RNA-Seq experiments with cells, in which these factors have been depleted, as well as a CASC3 knockout cell line. Conventional Illumina sequencing will be combined with single molecule RNA sequencing/single molecule PCR cDNA sequencing on the nanopore platform (Oxford Nanopore Technologies). In addition, we want to understand the importance of CASC3 on mRNA stability and mRNA translation using transcriptome-wide SLAM-Seq and Ribo-Seq, respectively. The combination of these techniques will enable us to better understand the various cellular functions of the EJC.
DFG Programme
Research Grants
