Project Area B: "Processing looks for Modifications"

The projects in Area B will build on deep, process-specific expertise in distinct RNA processing pathways and will investigate how RNA modifications influence these events. Individual projects will focus on defined processing steps, including splicing (B01), RNA surveillance and degradation mediated by the MTREC complex (B03), piRNA biogenesis (B04), rRNA biogenesis and ribosome assembly (B07), and mRNA decay in P-bodies (B08). In each case, prior evidence will motivate the hypothesis that RNA modifications are functionally coupled to the respective processing mechanism.
Using advanced approaches such as cryo-electron microscopy, structural biology, iCLIP, GLORI-Seq, and innovative long-read nanopore sequencing, the projects will dissect RNA recognition, quality control, and maturation at high resolution. For example, studies in B03 will elucidate how the MTREC complex selectively recruits RNA substrates for degradation, while B04 will explore how RNA cap methylation influences piRNA 5′-end maturation. In B07, new insights into rRNA processing and modification will be generated, including how dynamic modification patterns contribute to ribosome stability and function. B08 will examine how m6A modifications affect the stability of coding and noncoding mRNAs during decay in P-bodies.
Together, the Area B projects will create strong conceptual and methodological synergies, advancing a unified understanding of the interplay between RNA processing and modification.

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

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.