Neurons growing in a microfluidics device

The Murn Lab

For Creative Research of RNA Biology
INSPIRATION

Control of gene expression at the RNA level converts to life much of the information stored in our genomes. We are interested in understanding how RNA-linked processes allow cells to make decisions, respond to the environment, and communicate with one another, as well as how misregulation of these processes leads to cellular dysfunction and disease.

 

We pursue these problems in two major biological settings. First, we study the processing of protein-coding RNA, especially post-transcriptional regulation by mRNA-binding proteins (RBPs). We are particularly interested in understanding the formation and function of RBP-containing ribonucleoprotein complexes during early neuronal development and in neurodegeneration. This includes RBPs that are writers, readers, and erasers of RNA modifications, whose roles in neuronal development are largely unknown.

Second, we investigate the biogenesis of non-coding RNA as a major contributor to the total cellular RNA pool. Here, we are focusing on RNase P, one of only two multiple turnover ribozymes (the other one is the ribosome) that are found in organisms from all kingdoms of life. We study the structure-function relationships of RNase P and its evolutionary descendant, RNase MRP, as well as how the activities of these ribozymes contribute to cell homeostasis.

 

Our studies make heavy use of genome-wide and computational approaches (including techniques such as iCLIP, ribosome profiling, pseudoU-seq) in combination with traditional biochemistry and genetics to allow for comprehensive description of RNA networks and their regulating RBPs. We are integrating these approaches with in vitro stem cell differentiation and use of transgenic organisms to understand how control of gene expression at the RNA-level contributes to development and disease.

Some of our recent stories
  • A molecular program that determines cell shape

Cell shape is one of the most distinctive features of all somatic cells yet little is known about how it is specified. We discovered a molecular program, controlled at the RNA level, that determines the early shape of neurons. We found that a single molecule, a deeply conserved RNA-binding protein, called Unkempt, coordinates an entire morphology program to establish the early bipolar shape of neurons during embryonic development of an organism. Intriguingly, we found that this program also has the capacity to confer a similar morphology to cells of different lineages.

To understand what Unkempt-regulated processes might contribute to remodeling of cellular architecture, we first used iCLIP to define the identities and precise binding sites of Unkempt on its targeted RNA molecules. We found that Unkempt functions as a sequence-specific RNA-binding protein that targets coding regions of a defined set of ubiquitously expressed messages linked to protein metabolism and regulation of the cytoskeleton. RNA binding is required for Unkempt-induced remodeling of cellular shape and, as revealed by ribosome profiling, is directly coupled to a reduced production of the encoded proteins. Thus, during embryonic development, Unkempt controls a translationally regulated cell morphology program to ensensure proper structuring of the nervous system. These findings link post-transcriptional regulation of gene expression with cellular shape and have general implications for the development and disease of multicellular organisms. See the paper here.

Our work on the cover page at Genes and Development
  • Neuronal morphogenesis via a novel mode of RNA sequence recognition

The CCCH zinc finger proteins constitute the second largest group of RNA-binding proteins in mammals, yet their biology remains poorly understood. Following up on our initial description of Unkempt protein in specifying the early neuronal shape         , we wondered how the six CCCH zinc fingers of Unkempt - the largest such array of any mammalian protein - operate to recognize a relatively short stretch of RNA sequence. Structural and functional studies revealed the six zinc fingers form two compact RNA-binding folds, each consisting of three zinc fingers, with no similarity to any other structure in the annotated proteomes. We found that each zinc finger cluster functions as a separate RNA-binding unit that recognizes a distinct trinucleotide RNA motif, either UAG or a U-rich motif. Presence of both motifs and their relative position within coding regions of targeted genes is critical for high-affinity RNA binding, which, in turn, is required for translational repression of Unkempt-target genes, as well as for the induction of bipolar neuronal morphology.

Abstraction of neuronal morphogenesis via RNA binding

The evolutionary origins of the compact CCCH zinc finger clusters of Unkempt can be traced back roughly 600 million years to the emergence of metazoans and the evolution of the neuronal lineage. We found that RNA sequence specificity of Unkempt orthologs as well as their capacity to polarize cells have also remained conserved, suggesting an evolutionary link between the CCCH clusters of Unkempt and neuronal morphology. Given the high abundance of CCCH-type RNA-binding proteins and their wide functional diversity in different organisms, it will be important to determine the prevalence of such clusters, the rules that predict their formation and the set of properties beyond RNA binding that they may impart to proteins. We hope that our work would stimulate and provide some guidance to these endeavors.

  • Protein methylation in regulation of gene expression

Post-translational modifications (PTMs) of proteins regulate almost every aspect of cell biology by altering the functional properties of proteins, commonly in a reversible manner and at a relatively low energetic cost. Protein methylation is widespread and is perhaps one of the most functionally versatile forms of PTMs, with the capacity to impinge on essentially any cellular process. Accordingly, deregulation of protein methylation has been associated with a plethora of human disorders. We reviewed the field of protein methylation, paying particular attention to its established impact on gene transcription via histone methylation, as well as its less understood effects on post-transcriptional processing via methylation of RNA-binding proteins (see the paper here          ). Curiously, a substantial number of methylated sites in RNA-binding proteins appear to be buried deep within protein structure where they are unlikely to be accessible to methyl reader proteins. Buried methylated sites are most commonly located in the immediate vicinity of the bound RNA, which suggests that protein methylation could directly affect protein–RNA interactions. However, the significance of these methylation events remains largely unexplored.

Milestones in protein methylation research.