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

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

In eukaryotic cells, RNAs arising from pervasive transcription are rapidly degraded in the nucleus by the RNA exosome, aided by conserved adaptor complexes, such as the MTREC (Mtl1-Red1 core) complex. Here, the structure and function of MTREC will be studied by crystallography and cryo-EM. Structure and interplay with RNA substrates will be determined at the 5’-cap and the poly(A) tail, with a focus on effects of poly(A) polymerase Pla1 on poly(A) tail length. Furthermore, whether/which specific post-transcriptional RNA modifications lead to recruitment of MTREC will be investigated.

B04

In the germ cells of most animals, small RNAs called piRNAs interact with proteins from the Piwi-sub-family of Argonaute proteins to protect the genome from excessive activity of transposable elements. Here, the role of 5’ and 3’ terminal RNA modifications in piRNA precursor selection and processing will be studied in C. elegans with Piwi protein PRG-1. A method for piRNA precursor isolation will be developed to identify additional RNA modifications on these, and study their role in distinguishing piRNA from other RNA processing reactions. Finally, the function of the newly identified CEY-1 in piRNA precursor processing will be studied.

B05

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

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.