Our group has a long-standing interest in RNA metabolism, with a particular focus on the molecular mechanisms of eukaryotic RNA transport and degradation.
The degradation of RNA has emerged as a key step in the regulation of eukaryotic gene expression. In the case of mRNAs, the modulation of the transcript half-life is a powerful and versatile mechanism to swiftly alter the expression of proteins in response to changes in physiological conditions. The decay of mRNAs is performed by a set of macromolecular complexes that act in a sequential and coordinated manner, progressively eroding the ends of the transcript until its degradation is complete. These macromolecular assemblies contain only a few catalytically active subunits and a large number of regulatory components (Figure 1). The same enzymatic activities that are at play in constitutive RNA turnover are also involved in quality control pathways. Quality control pathways detect aberrant RNAs and bypass the normal rate-limiting steps, prompting fast decay. We are studying the macromolecular complexes that are involved in constitutive and regulated decay pathways with the aim to understand how they function at the atomic level and how they communicate with each other. To this end, we use a variety of biochemical, structural and functional approaches.
For the complete list of publications, see: https://www.biochem.mpg.de/conti/publications
RNA degradation: destroying the RNA body
A major pathway for the degradation of all kinds of RNAs involves a multiprotein assembly, the RNA exosome. Exosome-like complexes are present in all domains of life, highlighting the importance of this ancient machinery. After working on the prokaryotic complexes, we now focus entirely on the structure and mechanisms of the eukaryotic exosome (Figure 1, in green), using mainly S. cerevisiae as a model organism. The eukaryotic exosome cleaves the body of the RNA substrate from the accessible 3’ end, one nucleotide at a time, via a hydrolytic mechanism. In the case of RNA turnover and quality control pathways, exosome-mediated degradation proceeds processively until the transcript is eliminated. In nuclear RNA processing pathways, the degradation of the transcript is only partial and leads to the precise trimming of the RNA precursor to the appropriate functional form. In the past few years, we have characterized and visualized the first catalytically competent eukaryotic exosome core complex with an RNA trapped in a pre-catalytic state, revealing how the complex encages the ribonucleotide chain for processive degradation. Remarkably, although the chemistry of the reaction has diverged from prokaryotes to eukaryotes, the RNA-binding properties of the exosome cage have been largely conserved through evolution. We are now addressing how the exosome core complex is regulated by the cytoplasmic Ski complex (Ski2-Ski3-Ski8) and by the nuclear cofactors (Rrp6-Rrp47, Mpp6 and Mtr4). We are studying how RNA unwinding by the Ski2 and Mtr4 helicases is coupled to the processive ribonuclease activity of the exosome core. We are also interested in understanding how the two exoribonucleases of the nuclear exosome communicate with each other, with the aim to understand what determines whether a given RNA substrate is eliminated or partially trimmed.
RNA degradation: eroding the RNA extremities
Eukaryotic mRNAs are generally protected from degradation by specialized structures at their extremities: the poly-A tail at the 3’ end and the m7G cap at the 5’ end. Removal of these protective structures and the proteins bound to them precedes exosome-mediated degradation and is indeed the first and rate-limiting step in mRNA turnover. We are addressing how the poly-A tail is shortened. In the past years, we have been dissecting the multidomain architecture of two deadenylases, the Ccr4-Not complex (Figure 1, in pink) and the Pan2-Pan3 complex. We are now assembling the holo-complexes with the aim of understanding how the different portions of the complex work in the context of the messenger ribonucleoprotein particle (mRNP) and how they recruit peripheral proteins (such as the helicase Dhh1/DDX6). The Dhh1 helicase also binds Pat1, a factor that bridges the 3’ and 5’ ends of the mRNA (the Lsm1-7 and the decapping complexes) (Figure 1, in blue). We have studied how yeast Dhh1 binds Pat1 and how Pat1 in turns binds the Lsm1-7 complex. How these interactions interface with the decapping complex is an unresolved question. Our final goal is to use the S. cerevisiae proteins and complexes that we are reconstituting for the structural analysis to recapitulate the complex behavior of a eukaryotic mRNA decay pathway in vitro.
RNA quality control: eliminating aberrant mRNAs
Numerous surveillance pathways are in place at virtually all steps of RNA metabolism to detect errors. Nonsense-mediated mRNA decay (NMD) is the eukaryotic pathway that surveys mRNAs for the presence of premature stop codons (PTCs). PTC-containing mRNAs occur frequently in human cells and are promptly degraded, avoiding the synthesis of truncated and potentially harmful proteins. NMD is a cytoplasmic process that depends on translating ribosomes and, in the case of human cells, on the exon-junction complex (EJC). We have been working for several years to unravel the molecular mechanisms of NMD, in particular focusing on the EJC and the human NMD factors, the UPF and SMG proteins. In the past few years, we have addressed how the EJC recruits the conserved UPF proteins and how these in turn recruit the downstream effectors (the SMG5-7 proteins). We are now trying to understand how the SMG proteins orchestrate the interactions that bring together the UPF and EJC complexes to the stalled ribosome and induce fast decay of the aberrant mRNA.