Post-transcriptional networks & RNA-binding hubs Alexandre Smirnov

Post-transcriptional gene expression control is ubiquitous. It connects the transcriptional input from genes to the translational output in the form of proteins, in the end leading to the manifestation of phenotypes and adaptation.

Our research is focused on post-transcriptional mechanisms used by relatively simple bacterial-type genetic systems (bacteria and their distant descendants, mitochondria) to globally shape - and eventually adjust - their gene expression.

We are particularly interested in biology of highly conserved pleiotropic RNA-binding proteins (RBPs), noncoding RNAs, and the ribonucleoprotein particles (RNPs) they make up together.

By using and developing a wide variety of methods – from biochemistry to systems-level approaches and experimental evolution – we aim at understanding how these fascinating regulators work at all levels (from elementary molecular acts to their physiological consequences) and why they are so deeply conserved across large phylogenetic clades. We are also interested in theoretical aspects of RNA-binding hubs.

Grad-seq uses biochemical separation of cellular complexes (e.g. glycerol gradient) coupled to global mass spectrometry & RNA-seq to profile all protein & RNA components of the cell. Their in-gradient distributions help to visualise the biochemical diversity of cellular RNPs, predict their functions, and even identify new RNA classes & globally acting RBPs (RNA-binding hubs).

Our genome-wide approaches for transcriptomics and complexomics  Grad-seq

We use and develop ourselves original global techniques combining classical biochemistry and deep sequencing. Such approaches as Grad-seq enable comprehensive profiling of cellular transcripts and their protein partners, providing critical insights into their functions and the physical organisation of RNP-based post-transcriptional networks. Grad-seq, extensively used in bacteria to discover new classes of noncoding RNAs associated with conserved globally acting RBPs, such as Hfq, ProQ, and CsrA, is now being applied by our group to study the deeply diverged and poorly understood universe of mitochondrial ribonucleoproteins.

 CoLoC-seq

CoLoC-seq combines classical enzymatic kinetics of RNase digestion, deep sequencing, and mathematical modelling to determine which RNAs are genuinely present inside an organelle of interest (e.g. mitochondria) and which are merely surface-attached contaminants or RNase-resistant "impostors".

We are also interested in the amazing but technically challenging field of subcellular and organelle transcriptomics. Our recently developed CoLoC-seq approach enables robust, false positive-aware profiling of RNAs present inside membrane-bounded organelles, extracellular vesicles, and viral particles. Applied to human mitochondria, CoLoC-seq successfully and specifically captured mitochondrial DNA-encoded RNAs. Excitingly, it also detected a few small nuclear genome-encoded transcripts (Y RNAs, SNAR-A RNAs, tRNAs) in the mitochondrial intermembrane space. However, the extent of this "invasion" of the mitochondrial "territory" by nuclear RNAs turned out to be very low. CoLoC-seq is an ideal tool to dissect complex and potentially chimeric transcriptomes of genome-containing organelles (mitochondria, chloroplasts, apicoplasts) which, in many eukaryotic groups (e.g. plants, kinetoplastids, alveolates), evolved the ability to import and use select nuclear-encoded RNAs to sustain their own translation.

Check out a detailed protocol, examples of data analysis, & troubleshooting here.

Ribosome assembly  YbeY biology

In human mitochondria, YBEY, in complex with p32/C1QBP, recruits the ribosomal protein uS11m & chaperones its incorporation into the nascent small subunit of the mitochondrial ribosome. Disruption of this essential pathway results in unstable, translation-incompetent mitoribosomes & loss of cellular respiration.

Some of the most deeply conserved RBPs with the most profound impact on cellular physiology are found among the ribosomal proteins and ribosome biogenesis factors. Those are usually shared by nearly all representatives of the principal domains of Life (Bacteria, Archaea, Eukarya). The loss of such proteins is most often lethal or, at best, highly debilitating for the cell. We are primarily interested in ultraconserved assembly factors working on bacterial-type ribosomes, such as YbeY, which is found in nearly all bacteria and the eukaryotic organelles of bacterial origin (mitochondria and plastids). By applying a wide variety of approaches, we study the biology of such proteins at all levels, from their structure and most intimate molecular workings to their physiological impact at the level of entire human and bacterial cells. In the context of human mitochondria, such proteins as YBEY and its partners p32/C1QBP, ERAL1 and ribosomal protein uS11 play especially important roles as disease-related factors. Their mutations or deregulated accumulation result in severe mitochondrial diseases or cancer.

RNA-binding hubs in evolution

The pervasive impact of many globally acting RBPs on organismal fitness is well-known, and the scope of the post-transcriptional regulatory networks they control is getting well-understood. By contrast, the role they play in the evolution of their host cells is much less clear. Although their deletion is usually associated with pleotropic phenotypes, precluding survival of such mutants in nature, the secondary loss of some central RBPs, such as Hfq, Argonaute, or YbeY did happen in a few evolutionarily successful phyla maintaining rich post-transcriptional programmes. We want to understand whether and how a cell, which is used to employ such RBPs as critical components of its gene expression programmes, can overcome this addiction, reshuffle its regulatory networks, and - in the long run - untap new, so-far unavailable evolutionary resources. To tackle this intriguing question, we use experimental laboratory evolution of E. coli bacteria deficient in one of their most important RBPs - Hfq, ProQ or YbeY. By this means, we can observe - in real time! - how such crippled bacteria are changing their genomes, constantly inventing new ways to restore their fitness without recurring to the missing RBP.