Project Area B: "Processing looks for Modifications"
Each of the projects in Area B were conceived from a position of intimate knowledge on selected processing events, such as splicing (B01), an adaptor complex selecting RNA substrates for degradation (B03), the newly identified PETISCO complex in piRNA biogenesis (B04) and quality surveillance in translation (B05). Clear indications of the implication of modifications in the respective processing event form the basis for a working hypothesis, or in-depth analyses of known interactions between processing and modification.
B01
project discontinued (research period 1)
N6-methyladenosine (m6A) affects almost every stage of mRNA metabolism, including alternative splicing, and its alteration is associated with various physiological defects and diseases. Here, all m6A-dependent splicing events and all m6A sites that affect splicing will be mapped. The mechanistic interplay of effector proteins and the role of cis-regulatory elements that influence m6A-mediated regulation will be evaluated and links to RNA-binding proteins tested. This work will use Drosophila and human cells to work out conservation and differences and will be extended to the potential roles of other RNA modifications in alternative splicing regulation.
B03
Molecular insights into the role of MTREC in ncRNA processing and degradation
Non-coding RNAs or un-/mis-spliced mRNAs are rapidly recognized by adaptor complexes and targeted to the nuclear exosome. Here, we continue our work on the structure and function of the MTREC (Mtl1-Red1 core) and CNM (Ctr1-Nrl1-Mtl1) adaptors with special focus on their RNA target recognition due to aberrant processing or modification. CNM recognizes stalled spliceosomes and opens a link to quality control. We use an integral structural biology approach including split-tag purification or in vitro reconstitution, crosslinking mass-spec and RNA-seq technologies, and cryo-EM and X-ray analyses.
B04
Schlafen-related nucleases in worms and man
This project will address the function of a family of nucleases that is poorly understood: Schlafen nucleases. In particular, their RNA processing activities in relation to RNA modifications and transposon control will be studied. In nematodes, the function of the recently identified piRNA processing enzyme PUCH will be dissected further, whereas in human and mouse cells the enzyme SLFNL1 will be analyzed. By studying their interactions with other proteins, with RNA and with organelles, we expect to uncover novel insights into the roles of RNA modifications and RNA processing, notably those in innate immunity and genome defense
B05
project discontinued (research period 1)
tRNAs are heavily modified to aid function in translation and the lack of various modifications leads to aberrations in translation and aggregation of resulting proteins. Co-translational quality control detects and resolves various translation defects, including ribosome collisions. Here, a link between both phenomena will be explored by determining whether lack of specific tRNA modifications leads to ribosome collision, whether translation defects caused by lack of tRNA modifications are sensed and resolved by co-translational quality control machinery, and finally, whether this (partially) counteracts resulting protein aggregation.
B06
project discontinued (research period 1)
Eukaryotic ribosomal and spliceosomal RNAs are modified using snoRNAs as guides. Pseudouridylation of these RNAs is performed by H/ACA complexes, which use the snoRNA as a scaffold to assemble. We are interested in the detailed mechanisms how these complexes assembly, and how they perform their specific RNA modification. We use a combination of biochemical and analytical tools together with single molecule spectroscopy to identify interactions, structural dynamics and catalytic steps that underly the modification process.
B07
Mechanistic studies on the interplay of rRNA modification and processing with ribosome biogenesis, structure and function
Ribosomal RNA (rRNA) is heavily modified during maturation, where modification and processing are intimately linked and rRNA modifications impact ribosome function. Here, the interdependencies of rRNA modification and processing events, ribosome structure and function will be addressed. To this end, we will combine sucrose-gradient or crosslinking based isolation of maturing, translating and collided ribosomes with Nanopore direct RNA sequencing and cryo-EM, to study ribosomes from budding yeast, comparing wildtype to hypomodification mutants lacking ribosome maturation factors or factor New1.
B08
The role of P-bodies in m6A-mediated RNA decay
N6-methyladenine (m6A) is the most abundant internal mRNA modification in humans, crucial for RNA stability and cellular function in health and disease. Recently, we identified a specific role for m6A sites in the coding sequence (CDS) and discovered a novel RNA decay pathway, CDS–m6A decay (CMD). This project integrates biochemical and molecular techniques to investigate m6A-mediated transcript regulation, aiming to uncover its mechanisms and implications in cancer development.
