Modification and regulation of coding and non-coding RNA pathways

The genetic blueprints of all cellular functions are stored in a highly complex DNA structure known as the genome. Genes giving rise to proteins are referred to as coding and are transcribed to mRNAs, which carry the genetic information to ribosomes for protein production. Novel sequencing and profiling techniques revealed that the majority of the human genome, however, is non-coding and generates various classes of non-coding RNAs. Gene expression is highly specific to cell types or developmental stages, for example, and thus, gene expression must be highly regulated in time and space. Such regulatory events not only include regulation of gene transcription but also processes as diverse as RNA processing, RNA half-lives or translation efficiency.
During the past years it became more and more apparent that regulation of gene expression is an essential process and various pathways evolved to establish correct and robust gene expression programs. Among these pathways, RNA binding proteins (RBPs) are key factors that interact with RNAs and regulate their functions at multiple steps. Furthermore, miRNAs interact with distinct mRNAs and inhibit their expression. Finally, it has recently been unraveled that the RNA itself can be modified leading to changes in expression levels. Methylation of the Adenine base (m6A) has been widely studied and many exciting new gene regulatory processes have been uncovered. The general aim of the ‘moreRNA’ project is to study the regulatory potential and the interplay between RBPs, miRNAs and m6A methylation.
Gene expression programs dramatically change when cells start to proliferate and form tumors. How these changes are regulated is only poorly understood and therefore our work will help to better understand the role of post-transcriptional gene regulation in the development of diseases such as cancer. This is an important problem for the entire society since many types of cancer are still not curable and detailed molecular knowledge is urgently needed.
The ‘moreRNA’ project proposal analyzed two fundamental gene regulatory pathways. Our main objectives are: (i) regulation of gene expression by microRNAs (miRNAs) and the interplay with RBPs in cancer cell lines and tissue and (ii) deciphering the molecular principles of mRNA modification pathways and its biological functions.
We successfully identified more than 150 RBPs that can influence miRNA maturation and thus gene expression. We hypothesize that miRNA expression is controlled at a post-transcriptional level and this process is specific to cell types and also mis-regulated in cancer. Furthermore, we have characterized a number of these RBPs and assigned cellular functions to them. For example, LARP7 is a special RBP that can bind two RNA species simultaneously and thus tightly connects them for further maturation. In addition, METTL8 is a novel RNA methyltransferase that modifies a specific class of RNAs, referred to as transfer RNAs or tRNAs in mitochondria of human cells. This process is mis-regulated in pancreatic cancer and affects the activity of the respiratory chain which is located in the inner mitochondrial membrane.
Taken together, the moreRNA project contributed to our understanding of post-transcriptional regulation of miRNA expression and assigned functions to a number of RBPs. These findings are relevant for disease such as the Alazami syndrome or various types of cancer.

As initially planned, we have reached most of our scientific goals. Some objectives were starting points for long-term projects and they will be in the center of our research in the coming years.
We have performed a large biochemical screen to identify RBPs that interact with miRNA precursors and regulate their expression. This work has been carried out in a large panel of cancer cell lines and we found many cell line-specific interactions suggesting that these regulatory principles could be relevant for cancer development and progression. We have assigned cellular functions to several of these RBPs. Furthermore, we have started to characterize the protein-RNA complexes that are responsible for m6A modification in human cells. We have precisely mapped the molecular interactions of the METTL3-METTL14-WTAP complex and identified a number of contacts that are essential for the enzymatic activities. We have now continued to characterize this complex. We succeeded to stably produce a complex composed of four protein components and performed structural analyses using cryoEM. Although we are not yet at an atomic resolution, we hope to unravel the structural and molecular principles of this enzyme complex. Structural information may also help developing inhibitors of this enzyme complex, which could be important for disease treatment. We also found that YTH-proteins, which are known ‘readers’ of m6A are strongly associated with cell proliferation and downregulation is required for differentiation. These proteins are highly promising targets for cancer treatment and this is one of our long-term goal beyond the scope of the moreRNA project.

Our work on RBPs regulating miRNA expression has received a lot of attention and underscores the role of RBPs in miRNA processing. Our work opened a new direction that is now studied more actively by many other labs. We hope that our project will ultimately lead to new developments probably even towards clinical application addressing the molecular interplay between RBPs and their specific substrate RNAs in cancer including the highly cancer-relevant YTH proteins. RNA therapeutics is now with the COVID vaccination very present and RNA medicine will become one of the major directions in drug development. We hope that the moreRNA projects as significantly contributed and provided solid grounds for such developments.
In addition, we hope to provide detailed structural information on the m6A-generating enzyme complex. This is a highly competitive and tremendously active field of research at the moment and in case of successful completion of our work, we will generate highly visible results helping to move the entire field of RNA modification forward.

Periodic Reporting for period 4 - moreRNA (Modification and regulation of coding and non-coding RNA pathways)

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