Regulation of human spliceosome activation

• Applicant: Dr. Tonio Schütze
• Subject Area: General Genetics and Functional Genome Biology ; Biochemistry ; Cell Biology
• Term: from 2016 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 324935866

Protein-coding genes of higher eukaryotes typically contain non-coding intervening sequences (introns) flanked by coding regions (exons). Splicing, the excision of introns and simultaneous ligation of exons, is catalyzed by the spliceosome, which consists of preformed units called small nuclear ribonucleoprotein particles (snRNPs) and over 100 additional proteins. The assembly of the spliceosome happens de novo on every intron after the spliceosome recognizes short consensus sequences.Alternative splicing (AS), which is the differential usage of a particular splice sites, is the rule rather than the exception for most protein coding genes in humans. More than 90% of human genes show evidence of AS and cells must be able to change the selection of the right splice site in response to cellular needs. Constitutive exons, which are used in every transcript of a gene, have stronger splice sites whereas alternative exons have weaker splice sites. There are only very subtle differences between substrates with optimal and suboptimal splice sites. Consequently, the spliceosome has developed proofreading mechanisms that are necessary to sort substrates. Initial binding of the spliceosome to possible splice sites on a substrate is only the first of many steps, that lead to commitment to the splicing pathway.Defective splicing, for example caused by cancer mutations of core components of the splicing machinery, can lead to global changes in alternative splicing patterns. The molecular mechanism for this phenomenon is not well understood. My hypothesis is that an important component of splice site selection in humans is determined by proteins that are specific to complex B (B-specific proteins). In this spliceosome intermediate activation takes place and B complex RNA-dependent ATPases (Prp28, Brr2 and Prp2) are involved in the quality control of this selection. This is especially important in the context of weak splice sites involved in AS.In order to test the hypothesis, I will use Next-Generation Sequencing (NGS) to examine the splicing patterns of cellular RNA when protein expression of B-specific proteins or RNA-dependent ATPases are modified. It is an advantage that I will also use state-of-the-art Nanopore sequencing as this provides longer reads to identify known and novel splicing isoforms of genes. By comparison of the different resulting global splicing phenotypes in combination with an in vitro splicing system, I will deduce a hierarchy of events for these late stages of splicing.These experiments will address one of the big open questions in the field, which is to understand how the spliceosome ensures the high specificity of splice site selection. A better understanding of the characteristics of splicing decisions and the underlying spliceosome mechanism will not only result in a vast gain of knowledge but has also tremendous medical potential as estimates link 25% of all human diseases to mutations resulting in aberrant splicing.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professorin Dr. Melissa Jurica