Project

Discipline

base editing; CRISPR; splicing mechanism; spliceosome

Lead
Das Spliceosom katalysiert einen wichtigen Schritt der Verarbeitung der Ribonukleinsäure in höheren Eukaryoten. Wie seine Substrate ausgewählt werden, und auf welche Weise die Genauigkeit des Prozesses gewährleistet wird, ist für das menschliche Spliceosom noch nicht erschlossen. Dieses Projekt soll sich dieser Fragenstellung widmen.
Lay summary
Inhalt und Ziele des Forschungsprojekts Das menschliche Genom kodiert ungefähr 20000 Gene. Im Durchschnitt werden diese Gene durch acht Introne geteil, welche mittels Splicing entfernt werden. Das Splicing wird durch das Spliceosom katalysiert. Paradoxerweise können sich Intronsignale für das Splicing stark unterscheiden und trotzdem wird das Splicing sehr präzise durchgeführt.  Um Mechanismen dieser Spezifität des Spliceosoms zu entschlüsseln, habe ich ein System aufgesetzt, welches mittels CRISPR Technologie mir erlaubt programmierte Mutagenese auf die Proteine des Spliceosoms anzuwenden. Das Projekt untersucht in einem ersten Schritt wie Mutationen Resistenzen gegenüber Splicinginhibitoren hervorrufen können. In einem weiteren Schritt soll mittels Reportern untersucht werden, wie die Genauigkeit des Splicings gegeben wird.   Wissenschaftlicher und gesellschaftlicher Kontext des Forschungsprojekts Viele, vor allem vererbbare Krankheiten, haben ihren Ursprung in einem Defekt im Splicing des betroffenen Genes. Obwohl einige dieser Krankheiten therapierbar sind, bleiben aber viele andere nicht behandelbar. Ein grundlegendes Verständnis dieses Prozesses bleibt daher unabdingbar. Mein Ansatz, das Spliceosom unvoreingenommen und gesamtheitlich zu untersuchen, kann daher dazu beitragen diesen Prozess besser zu verstehen und die Basis zur Erforschung weiterer Behandlungsansätze liefern.

Direct link to Lay Summary

Last update: 12.07.2021

Number	Title	Start	Funding scheme

Rationale: The human genome encodes approximately 20,000 protein-coding genes. On average, genes are split by eight introns, and a single gene can harbour three hundred introns. The spliceosome catalyses the essential mRNA processing step of intron removal. While the majority of the spliceosome’s components are highly conserved from yeast to human, this is not the case for the spliceosome’s substrates. This raises a central question: How can a conserved machine accomplish with precision the same reaction in the yeast S. cerevisiae, in which strict adherence to consensus sequences are required for the process, and in human cells, where intron signals for splicing are highly variable?Although this question may be partially answered by involvement of auxiliary factors in humans, recent structures of the spliceosome raise another untested hypothesis, namely that the core proteins of the spliceosome have evolved to accept more diverse substrates. Regardless of scenario, the key contributors within the human spliceosome enabling its tolerance for its broader target range (despite the requirement for fidelity) remain unknown. Addressing these key questions has been hampered by a lack of tools to interrogate individual residues of essential cellular machines in human cells. To have such a tool at hand, I have set-up a system for CRISPR/Cas9-base editing in human haploid cells during my SNF Early Postdoc.Mobility Fellowship. In an initial pilot screen this system has yielded promising results and can now be employed to query the spliceosome to gain insights and answers to these questions.Objective: I propose to decipher mechanisms of spliceosomal fidelity by combining programmed mutagenesis of the entire spliceosome using CRISPR base editing technology coupled with high-throughput splicing measurements. Aim 1: Understanding pladienolide B resistance and sensitivity. I performed a base editing screen and identified sgRNAs conferring resistance and sensitivity to the splicing inhibitory drug pladienolide B. In a next step, I will confirm targets and describe the mutations. In addition to demonstrating the feasibility of our screening approach, mutants found outside the known pladienolide B target spectrum will further our understanding of major steps in splicing such as branchpoint and 3’ splice site recognition as well as escape from splicing inhibition.Aim 2: Uncovering the mechanism of spliceosome fidelity in human cells via global mutagenesis. I will generate a panel of haploid cell lines carrying bichromatic splice reporters, which will carry disease-relevant mutations allowing me to query both 5’ splice site and 3’ splice site definition, as well as splicing fidelity. These cell lines will be transduced with my sgRNA library and flow cytometry will be used to sort cells based on changes in splicing efficiency over time. Sequencing of the guides from the sorted cells, will reveal mutable spliceosomal residues that determine acceptance versus rejection of each splicing substrate.Significance: I propose to elucidate the basis of human splicing fidelity and processing at the single amino acid level in the core spliceosome and peripheral spliceosome proteins. This approach will illuminate the activities of the human spliceosome at an unprecedented level. Splicing is frequently perturbed in human disease. A better understanding of individual spliceosomal components, especially at the amino acid level, may enable targeted drug discovery approaches. Concomitantly, my project aims to illuminate mechanisms that operate at the heart of the human spliceosome, a remarkable molecular machine.