General Biology of RNA and Proteins 3900-BT1-1
Lecture 1 — Introduction to the chemistry and structural dynamics of RNA. Nucleotide chemistry: 2′-OH reactivity, phosphodiester hydrolysis, tautomerism and protonation, metal-ion coordination. Secondary and tertiary structure hierarchy: helices, loops, bulges, pseudoknots, A-minor motifs, kissing loops, G-quadruplexes. Thermodynamics and kinetics of folding; misfolded traps and chaperone-assisted folding. Conformational ensembles: excited states, transient base pairs, ligand- and riboswitch-coupled conformational capture. Methods overview: NMR relaxation dispersion, SAXS, cryo-EM, SHAPE/DMS chemical probing and transcriptome-wide structure mapping, single-molecule FRET.
Lecture 2 — Cap chemistry. Co-transcriptional capping: RNA triphosphatase, guanylyltransferase, N7-methyltransferase; the m7G cap and 2′-O-methylated cap1/cap2. Cap-binding proteins — CBC (CBP20/80) in the nucleus, eIF4E in translation initiation — and the cap as an identity tag for "self" RNA. Cap-dependent quality control: decapping (DCP2, DXO, Nudt16), non-canonical caps (NAD⁺, FAD, dpCoA), 5′-end surveillance. Viral cap mimicry and cap snatching; cap analogues (ARCA, CleanCap-type structures) and their role in mRNA vaccine manufacture and innate-immune silence.
Lecture 3 — RNA life cycle: stability and degradation. Determinants of mRNA half-life: 3′UTR elements (AREs, CDEs), codon optimality, poly(A) tail length and deadenylation by PAN2–PAN3 and CCR4–NOT. Decapping and 5′→3′ decay by XRN1; 3′→5′ decay by the exosome and SKI complex. Endonucleolytic pathways: RNase L, SMG6, Regnase/Roquin. Co-translational and surveillance decay: NMD, no-go and non-stop decay. Phase-separated compartments — P bodies and stress granules — and whether they are causes or consequences of repression. Measuring decay: metabolic labelling (SLAM-seq, TT-seq), transcription shut-off, nascent-chain profiling.
Lecture 4 — Epitranscriptomics: RNA editing and chemical modifications. The modification landscape: m6A, m5C, hm5C, ac4C, pseudouridine, 2′-O-methylation, m1A, inosine. Writers, erasers and readers — METTL3/METTL14/WTAP, FTO and ALKBH5, YTH-domain proteins — and the contested reversibility of marks. Consequences for splicing, export, translation, decay and phase behaviour. A-to-I editing by ADAR1/ADAR2: recoding versus self/non-self discrimination, and the interferon link. C-to-U editing by APOBECs. Detection methods and their pitfalls: antibody-based mapping, chemical/enzymatic single-base methods (GLORI, bisulfite, RBS-seq), direct nanopore sequencing; stoichiometry versus site inventories.
Lecture 5 — Protein structure and biogenesis. From sequence to fold: hierarchy, energy landscapes, folding funnels, and why the folding problem and the structure-prediction problem are not the same. Co-translational folding, ribosome-associated chaperones (trigger factor, Hsp70/Hsp40), chaperonins (GroEL, TRiC/CCT), Hsp90 clients. Proteostasis network and quality control: UPR, heat-shock response, aggregation and amyloid. Intrinsically disordered regions, low-complexity domains and condensate formation. Structure determination and prediction: X-ray crystallography, cryo-EM, NMR, AlphaFold-class models — confidence metrics, limitations and appropriate use.
Lecture 6 — Protein sorting and export. Signal sequences and the sorting decision tree. Co- and post-translational targeting to the ER: SRP, Sec61 translocon, SecA/SecYEG in bacteria; membrane protein insertion (YidC/EMC, GET pathway). N-glycosylation, disulfide formation, ERAD and the calnexin/calreticulin cycle. Vesicular traffic: COPII budding and cargo selection, COPI retrieval, Golgi maturation, clathrin and adaptor-mediated sorting, tethering and SNARE-mediated fusion. Alternative routes: mitochondrial and peroxisomal import, nuclear transport, unconventional secretion, extracellular vesicles. Bacterial secretion systems and their exploitation by pathogens.
Lecture 7 — Regulation of protein stability in cells and in vivo. The ubiquitin system: E1–E2–E3 architecture, chain linkage types and the ubiquitin code; deubiquitinases; UBL modifiers (SUMO, NEDD8). The 26S proteasome: recognition, unfolding, processivity, degrons and N-/C-degron pathways. Autophagy and lysosomal degradation; selective autophagy receptors. Measuring stability: pulse-chase, SILAC turnover proteomics, GPS/degron reporters, tandem fluorescent timers, in vivo half-life measurement. Induced degradation as a therapeutic modality and research tool: PROTACs, molecular glues, auxin/dTAG conditional degron systems.
Lecture 8 — mRNA localisation, transport and subcellular dynamics. Why localise an mRNA: efficiency, cell polarity, local proteome control, avoiding ectopic activity. Zipcodes and trans-acting factors; motor-driven transport on microtubules and actin; anchoring and localised translational activation; diffusion-and-capture versus directed transport; hitchhiking on organelles. Model systems: Drosophila oskar/bicoid/gurken, budding yeast ASH1, neuronal dendrites and axons, migrating fibroblasts, mitochondrial and ER surfaces. Imaging and mapping: MS2/PP7 tagging, single-molecule FISH, HCR, MERFISH/seqFISH, APEX- and proximity-based subcellular transcriptomics, live-cell translation imaging (SunTag).
Lecture 9 — Exploring the world of RNA-binding proteins. Classical RBD architecture (RRM, KH, dsRBD, zinc finger, PUF, Pumilio, cold-shock) versus unconventional and moonlighting RNA binders; the enigmatic RBP concept; RNA-binding by metabolic enzymes; the riboregulation hypothesis. System-wide discovery: RNA interactome capture, OOPS/XRNAX, RBDmap, CLIP family methods (HITS-CLIP, PAR-CLIP, iCLIP, eCLIP) and their controls and artefacts. Sequence versus structure specificity; multivalency, condensates and RNA as a scaffold. RBPs in disease: TDP-43, FUS, SMN, MSI2, repeat expansions.
Lecture 10 — Host–pathogen interactions in the context of RNA. RNA as a pathogen signature: RIG-I, MDA5, LGP2, PKR, OAS/RNase L, TLR3/7/8 — the chemical basis of self versus non-self (5′-ppp, duplex length, 2′-O-methylation, modification status). Interferon induction and the ISG effector repertoire (IFITs, ZAP, APOBEC3, ADAR1's paradoxical role). Viral countermeasures: cap mimicry, decoys, methylation, VSR proteins, nuclease evasion, hijacking of host RBPs and stress-granule antagonism. Coronaviruses as a worked case study: replication–transcription complexes, sgRNA synthesis, nsp1-mediated host shutoff. Bacterial and parasite sRNA-based regulation; RNA-based host–microbe signalling.
Lecture 11 — Peptides and proteins as immunomodulators. Host-defence/antimicrobial peptides: defensins, cathelicidins (LL-37), structural classes, membrane-selective mechanisms and immunomodulatory (non-microbicidal) activities — chemotaxis, wound repair, dampening of TLR responses. Cytokines and engineered cytokine variants; checkpoint-modulating peptides; peptide vaccines and epitope design; MHC presentation as peptide chemistry. Peptide drug development: proteolytic stability, cyclisation, D-amino acids and peptidomimetics, staples, PEGylation and lipidation, delivery and immunogenicity/selectivity liabilities. Regulatory and translational realities of peptide therapeutics.
Lecture 12 — RNA and protein research: future perspectives and directions. Where the field is going: programmable RNA medicines (mRNA vaccines, self-amplifying RNA, circular RNA, ASOs, siRNA, RNA editing and CRISPR–Cas13); RNA and protein design with deep learning and its evaluation; single-molecule and in situ structural biology; spatial multi-omics; condensate pharmacology; targeted degradation beyond the ubiquitin system. Cross-cutting issues: reproducibility and reporting standards for high-throughput RNA data, benchmark leakage in computational prediction, biosafety and dual-use considerations, and open data/FAIR practice. Student project presentations.
Course coordinators
Type of course
Mode
Prerequisites (description)
Learning outcomes
Knowledge and understanding
Upon completing the course, students will be able to:
1. Explain the structural, chemical and thermodynamic principles governing RNA folding, dynamics and conformational heterogeneity.
2. Describe co- and post-transcriptional processes that shape RNA molecules, including capping, epitranscriptomic modification, RNA editing and decay.
3. Describe the mechanisms of protein folding, quality control, intracellular and extracellular trafficking, and regulated degradation through the ubiquitin–proteasome system and autophagy.
4. Explain how RNA-binding proteins, mRNA localisation and RNA-based innate immune sensing regulate gene expression and host defence.
5. Describe the principal experimental and computational methods used to study RNA and protein structure, dynamics and interactions.
6. Relate molecular mechanisms of RNA and protein regulation to disease processes and therapeutic strategies, including mRNA vaccines, targeted protein degradation and RNA editing.
Skills
Upon completing the course, students will be able to:
1. Interpret data generated using cryo-EM, CLIP-seq, SHAPE/DMS chemical probing, pulse-chase labelling and single-molecule imaging.
2. Compare the strengths, limitations and potential artefacts of experimental and computational methods used in RNA and protein research.
3. Integrate structural, biochemical, imaging and computational data to evaluate molecular mechanisms.
4. Assess how experimental findings may inform the development or optimisation of RNA- and protein-based therapies.
Competences
Upon completing the course, students will be able to:
1. Critically evaluate scientific evidence concerning RNA and protein structure, dynamics and interactions.
2. Apply rigorous standards of reproducibility, documentation and reporting when assessing molecular life-science research.
3. Recognise and appropriately address biosafety, ethical and dual-use considerations in research involving RNA, proteins and related technologies.
4. Communicate scientific interpretations clearly and responsibly in academic and research settings.
Assessment criteria
The exam takes the form of a multiple-choice test covering the material presented during the lectures. It lasts up to 90 minutes and consists of up to 30 questions or tasks, each scored individually, for a maximum total of 100 points.
The final grade is based on the percentage of points obtained out of the maximum possible score:
- up to 50% – fail (2.0),
- 51–60% – satisfactory (3.0),
- 61–70% – satisfactory plus (3.5),
- 71–80% – good (4.0),
- 81–90% – good plus (4.5),
- 91–100% – very good (5.0).
A minimum of 51% of the total points is required to pass the course.
A maximum of 2 absences is allowed.
Bibliography
Recommended literature, however mandatory readings will be specified by the instructors during the course.
1. Ganser LR, Kelly ML, Herschlag D, Al-Hashimi HM. The roles of structural dynamics in the cellular functions of RNAs. *Nat Rev Mol Cell Biol.* 2019;20(8):474–489. doi:10.1038/s41580-019-0136-0. PMID: 31182864.
2. Mortimer SA, Kidwell MA, Doudna JA. Insights into RNA structure and function from genome-wide studies. *Nat Rev Genet.* 2014;15(7):469–479. doi:10.1038/nrg3681. PMID: 24821474.
3. Ramanathan A, Robb GB, Chan SH. mRNA capping: biological functions and applications. *Nucleic Acids Res.* 2016;44(16):7511–7526. doi:10.1093/nar/gkw551. PMID: 27317694.
4. Topisirovic I, Svitkin YV, Sonenberg N, Shatkin AJ. Cap and cap-binding proteins in the control of gene expression. *Wiley Interdiscip Rev RNA.* 2011;2(2):277–298. doi:10.1002/wrna.52. PMID: 21957010.
5. Garneau NL, Wilusz J, Wilusz CJ. The highways and byways of mRNA decay. *Nat Rev Mol Cell Biol.* 2007;8(2):113–126. doi:10.1038/nrm2104. PMID: 17245413.
6. Schoenberg DR, Maquat LE. Regulation of cytoplasmic mRNA decay. *Nat Rev Genet.* 2012;13(4):246–259. doi:10.1038/nrg3160. PMID: 22392217.
7. Roundtree IA, Evans ME, Pan T, He C. Dynamic RNA modifications in gene expression regulation. *Cell.* 2017;169(7):1187–1200. doi:10.1016/j.cell.2017.05.045. PMID: 28622506.
8. Nishikura K. Functions and regulation of RNA editing by ADAR deaminases. *Annu Rev Biochem.* 2010;79:321–349. doi:10.1146/annurev-biochem-060208-105251. PMID: 20192758.
9. Balchin D, Hayer-Hartl M, Hartl FU. In vivo aspects of protein folding and quality control. *Science.* 2016;353(6294):aac4354. doi:10.1126/science.aac4354. PMID: 27365453.
10. Kim YE, Hipp MS, Bracher A, Hayer-Hartl M, Hartl FU. Molecular chaperone functions in protein folding and proteostasis. *Annu Rev Biochem.* 2013;82:323–355. doi:10.1146/annurev-biochem-060208-092442. PMID: 23746257.
11. Rapoport TA. Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes. *Nature.* 2007;450(7170):663–669. doi:10.1038/nature06384. PMID: 18046402.
12. Zanetti G, Pahuja KB, Studer S, Shim S, Schekman R. COPII and the regulation of protein sorting in mammals. *Nat Cell Biol.* 2011;14(1):20–28. doi:10.1038/ncb2390. PMID: 22193160.
13. Komander D, Rape M. The ubiquitin code. *Annu Rev Biochem.* 2012;81:203–229. doi:10.1146/annurev-biochem-060310-170328. PMID: 22524316.
14. Burslem GM, Crews CM. Proteolysis-targeting chimeras as therapeutics and tools for biological discovery. *Cell.* 2020;181(1):102–114. doi:10.1016/j.cell.2019.11.031. PMID: 31955850.
15. Martin KC, Ephrussi A. mRNA localization: gene expression in the spatial dimension. *Cell.* 2009;136(4):719–730. doi:10.1016/j.cell.2009.01.044. PMID: 19239891.
16. Das S, Vera M, Gandin V, Singer RH, Tutucci E. Intracellular mRNA transport and localized translation. *Nat Rev Mol Cell Biol.* 2021;22(7):483–504. doi:10.1038/s41580-021-00356-8. PMID: 33837370.
17. Hentze MW, Castello A, Schwarzl T, Preiss T. A brave new world of RNA-binding proteins. *Nat Rev Mol Cell Biol.* 2018;19(5):327–341. doi:10.1038/nrm.2017.130. PMID: 29339797.
18. Gerstberger S, Hafner M, Tuschl T. A census of human RNA-binding proteins. *Nat Rev Genet.* 2014;15(12):829–845. doi:10.1038/nrg3813. PMID: 25365966.
19. V'kovski P, Kratzel A, Steiner S, Stalder H, Thiel V. Coronavirus biology and replication: implications for SARS-CoV-2. *Nat Rev Microbiol.* 2021;19(3):155–170. doi:10.1038/s41579-020-00468-6. PMID: 33116300.
20. Hur S. Double-stranded RNA sensors and modulators in innate immunity. *Annu Rev Immunol.* 2019;37:349–375. doi:10.1146/annurev-immunol-042718-041356. PMID: 30673536.
21. Mookherjee N, Anderson MA, Haagsman HP, Davidson DJ. Antimicrobial host defence peptides: functions and clinical potential. *Nat Rev Drug Discov.* 2020;19(5):311–332. doi:10.1038/s41573-019-0058-8. PMID: 32107480.
22. Hancock RE, Sahl HG. Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. *Nat Biotechnol.* 2006;24(12):1551–1557. doi:10.1038/nbt1267. PMID: 17160061.
23. Jumper J, Evans R, Pritzel A, et al. Highly accurate protein structure prediction with AlphaFold. *Nature.* 2021;596(7873):583–589. doi:10.1038/s41586-021-03819-2. PMID: 34265844.
24. Winkle M, El-Daly SM, Fabbri M, Calin GA. Noncoding RNA therapeutics — challenges and potential solutions. *Nat Rev Drug Discov.* 2021;20(8):629–651. doi:10.1038/s41573-021-00219-z. PMID: 34145432.
Suggested supplementary reading (optional, not examinable):
Zaccara S, Ries RJ, Jaffrey SR. Reading, writing and erasing mRNA methylation. *Nat Rev Mol Cell Biol.* 2019;20(10):608–624. doi:10.1038/s41580-019-0168-5. PMID: 31520073.
Knowles TPJ, Vendruscolo M, Dobson CM. The amyloid state and its association with protein misfolding diseases. *Nat Rev Mol Cell Biol.* 2014;15(6):384–396. doi:10.1038/nrm3810. PMID: 24854788.
Parker R, Sheth U. P bodies and the control of mRNA translation and degradation. *Mol Cell.* 2007;25(5):635–646. doi:10.1016/j.molcel.2007.02.011. PMID: 17349952.
Castello A, Fischer B, Eichelbaum K, et al. Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. *Cell.* 2012;149(6):1393–1406. doi:10.1016/j.cell.2012.04.031. PMID: 22658674.
Schoggins JW. Interferon-stimulated genes: what do they all do? *Annu Rev Virol.* 2019;6(1):567–584. doi:10.1146/annurev-virology-092818-015756. PMID: 31283436.
Muttenthaler M, King GF, Adams DJ, Alewood PF. Trends in peptide drug discovery. *Nat Rev Drug Discov.* 2021;20(4):309–325. doi:10.1038/s41573-020-00135-8. PMID: 33536635.