In our research we strive to understand the normal function and regulation of RNA processing, in particular alternative splicing, in myogenesis and how dysregulation contributes to muscle disease. After transcription, RNAs have to be spliced, capped and poly-adenylated, edited, exported from the nucleus, trafficked and translated. Each of these steps is heavily regulated, and additional pathways impact transcript stability. Regulation is achieved largely through the activity of RNA binding proteins, canonically identified to have at least one of a panel of characterized RNA binding domains. These processing steps determine the mRNAs that are available for translation, determining which proteins and protein isoforms are ultimately expressed by a cell.
There are many steps in the processing and development of an RNA before it can be translated.
Muscles rely on alternative splicing and RNA regulation to fine-tune their contractile properties. Expression of different isoforms of sarcomeric proteins, for example myosin, actin or titin, have been demonstrated to impact actomyosin contractility. During normal development and in response to exercise, different muscles display distinct patterns of splicing and protein isoform expression. These expression patterns are frequently disrupted in disease, resulting in expression of the wrong sarcomere protein isoforms and leading to structural defects, disruption of function, atrophy and in many cases fiber loss. Diseases such as dilated cardiomyopathy and myotonic dystrophy are caused by dysregulation of RNA binding proteins, and many other diseases are characterized by misregulation of splicing or RNA trafficking. Despite this, our understanding of RNA regulatory mechanisms as well as the proteins capable of modifying and regulating RNA is limited and incomplete.
Loss of splicing factors leads to defects in muscle development and function, often causing disease.
We investigate RNA regulation and muscle development using mainly Drosophila melanogaster as our model organism. In addition to the vast array of genetic tools and fast generation time, muscle proteins and structure are highly conserved from flies to vertebrates. In particular we focus on the indirect flight muscles (IFMs), which are distinct from other fly muscles with their asynchronous and stretch-activated contractions and lack of lateral alignment between myofibrils. The development of the IFMs is well characterized and stereotyped, allowing detailed investigation of basic mechanisms of sarcomere formation, myofibril organization and muscle maturation. Using this model, we hope to understand the mechanisms guiding tissue-specific alternative splicing and RNA regulation, as well as identify novel splicing regulators. Our results in Drosophila will be directly relevant to understanding mechanisms of pathogenesis in human muscle disease, as well as conserved mechanisms of muscle development and sarcomerogenesis.
A well-characterized series of morphological events defines flight muscle development.
Bruno1 (also called Bru1, Arrest, Aret) is a conserved member of the CELF-family of RNA binding proteins containing 3 RRM domains. The human homolog CELF1 is known to be mis-regulated in myotonic dystrophy, and together with MBNL to control many of the RNA regulatory processes disrupted in patient muscles. I previously showed that Bru1 regulates flight muscle specific alternative splicing in Drosophila, and its loss leads to defects in sarcomere growth, hypercontraction and ultimately muscle fiber loss. In a project funded by the Deutsche Forschungsgemeinschaft, we are investigating the mechanism by which Bru1 regulates splicing in flight muscle.
To understand developmental mechanisms leading to myofibril phenotypes in Bru1 mutants, we use genetics, confocal microscopy and transmission electron microscopy. We are further evaluating the function of different Bru1 isoforms, including an uncharacterized isoform containing only 2 RRMs. We also would like to identify Bru1 binding elements in direct RNA targets using the iCLIP technique. We expect to define regulatory elements in introns or exons near splice junctions regulating alternative splicing and possibly in UTR regions regulating mRNA localization, stability or translation. These studies will identify the developmental mechanisms misregulated in Bru1 mutants leading to defects in myofibril growth, assign specific functions to individual Bru1 isoforms and identify direct Bru1 targets in flight muscle. These findings may have vertebrate relevant implications, in particular for the function of CELF family members in muscle development and disease.
Bruno mutant fibers are lost due to sarcomere growth and hypercontraction defects.
CHERP (Calcium homeostasis endoplasmic reticulum protein) was first identified in vertebrate cell culture to play a role in cellular calcium homeostasis, but has since been shown to be an accessory component of the U2 spliceosomal complex. Together with Rbm17 and U2SURP it controls alternative splicing as well as suppressing cryptic splice sites. In certain cancers, its overexpression is sufficient to drive proliferation and suppress apoptosis, suggesting it can act as an oncogene. Our preliminary data implicates the Drosophila homolog of CHERP, Scaf6, in muscle development.
In collaboration with Dr. Rippei Hayashi from the Australian National University, we are working to understand Scaf6 function in Drosophila development. We are focused on characterizing mutant and RNAi knockdown phenotypes in muscle and other fly tissues. We hope to gain detailed mechanistic insight into Scaf6 function through RNA-Seq and mass spectrometry experiments. We are further investigating the evolutionary conservation of Scaf6/CHERP function in mouse myoblast C2C12 cells. Our results will provide insight into spliceosome function and the coordination of different splicing processes with relevance to myogenesis and the regulation of cell proliferation.
GFP-tagged fosmid lines allow monitoring of sarcomere protein levels in transgenic flies.
N6-methyladenosine (m6A) is a methyl modification found on adenine in some portion of mRNA molecules. Mettl3, together with Mettl14 and other components of the writer protein complex, deposit the methyl mark on target mRNAs. m6A modification has been suggested to regulate diverse steps in RNA processing from alternative splicing and localization to turnover and translation, although the cell-type specific function of m6A in muscle is largely unknown. Recent studies have demonstrated m6A modification is essential for sex-specific splicing in Drosophila embryos, and have identified key players in the m6A modification pathway. Interestingly, surviving adults are flightless, although this was suggested to be due to neuronal defects observed in Mettl3 mutants.
Given the prevalence of the m6A mark and the conservation of the modification pathway throughout evolution, we are exploring the role and requirement of m6A modification in muscle development and function in Drosophila. Using behavior and genetics, we are teasing apart the requirement for m6A modification in muscles and neurons as it contributes to adult behaviors including flight and climbing. We would further like to identify RNAs that are m6A modified in differentiated muscle cells, and assay how gene expression and alternative splicing are impacted in Mettl3 mutants using mRNA-Seq. Our data indicate important muscle-intrinsic functions of m6A, and will offer novel insights into mechanisms of RNA regulation in muscle development.
Behavior data plot showing flight ability of diverse Mettl3 genetic manipulations.
There are hundreds of proteins known to bind RNA, mostly containing one or more canonical RNA-binding domains. However, recent RNA-interactome capture studies have demonstrated that far more proteins than previously appreciated have the ability to bind RNA. The function of this binding is currently poorly characterized, as is the tissue-specificity of the proteins shown to bind RNA. We are interested in identifying proteins that bind RNA in muscle, potentially impacting alternative splicing, transport, stability, translation and other steps in RNA regulation in a cell-type specific manner.
We employ several different approaches to identify new RNA binding proteins and characterize their function in muscle. We identify tissue-specific candidates from genome-wide deep sequencing and RNAi screen datasets, as well as using biochemical purification and -omics technologies. We evaluate the muscle-specific function of candidates using RNAi and genetics approaches in vivo in flies, and in culture in C2C12 myoblast cells. We further plan to develop fluorescent reporters for defined muscle-type specific alternative splice events and use these to screen for factors controlling a defined set of alternative splice events, harnessing the power of forward genetics in Drosophila. Our data will identify novel RNA binding proteins, new pathways regulating RNA metabolism and tissue-type specific logic in splicing regulation.
Immunoprecipitation of CHERP from mouse myoblast C2C12 cells.