Molecular techniques of RNA analysis 1400-225ZMTA

Lectures:

1. "RNA world" part 1.
The RNA world concept. Nobel Prizes in the field of RNA. Catalytic RNA molecules - classes, mechanism, occurrence. SELEX. RNA world vestiges - ribosome, spliceosome, RNA viruses. Diversity of RNA classes in Eukaryotes and their metabolism. Basic mechanisms of gene expression regulation from transcription to translation. Alternative processes. RNA surveillance mechanisms. Ribonucleoprotein structures, cellular condensates.

2. "RNA world" part. 2.

3. "Reverse Transcription Techniques. Transcription techniques: TRO, ChIP, RIP and DIP. RNA polyA ends analysis".
Reverse Transcription (RT) – theory. Overview of RT-based techniques: RT-PCR including semi-quantitative, pulse RT-PCR, RACE, cRT-PCR, primer extension technique. Techniques for studying nascent transcripts: nuclear "Transcription Run-On", chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RiP). RNA Polymerase I transcription termination studies will illustrate the above techniques. Specificity of DNA binding by transcription factors: "ChIP on chip", DNA immunoprecipitation (DIP). Examination of polyA RNA ends (PASE technique, isolation of polyA+ RNA fraction).

4. "Non-coding RNA".
Non-coding RNA (ncRNA). Biogenesis and functions of small and long non-coding RNAs in Eukaryotic cells. Transcriptional and post-transcriptional silencing mechanisms dependent on ncRNAs. RNP complexes involved in silencing. The role of chromatin in the regulation of gene expression.

5. "RNA world" part. 3.

6. "Real-time PCR (quantitative; qPCR)".
The qPCR theory: methods of DNA detection, basics of primers design, hybridization probes, introduction to calculations. Applications: determination of the transcripts levels in the cell, detection of pathogen nucleic acids, detection of single-nucleotide polymorphisms (SNP). Good laboratory practice in qPCR experiments and the most common "pitfalls" waiting for experimenters.

7. "RNA in neurons".
mRNA transport in dendrites and axons, local translation in response to synaptic stimulation. Methods of mRNA visualization in living neuron in real time, in situ hybridization, biochemical methods for direct detection of transcripts undergoing local translation in neurons.

8. "RNA metabolism in physiological processes".
The role of RNA processes and ncRNAs in cell metabolism in plants and animals: hormone signaling, embryonic and generative development, circadian clock, resistance to stress and pathogens.

9. "Study of RNA metabolism enzymes on the example of the exosome".
RNA degradation pathways in eukaryotic organisms. Exo- and endonucleases. Exosome: a multifunctional and multicomponent complex. Structural and functional studies of the exosome in yeast and human cells. Mechanism of action, cooperation of exo- and endo-nucleolytic activity.

10. "RNA Structure vs. Function. In vitro RNA structure mapping. Methods of studying transcriptomes".
In vitro RNA structure mapping: molecular probes that specifically digest RNA structure and sequence. Riboswitches and aptamers – natural and selected for the needs of drugs or biosensors. High-throughput transcriptome testing methods based on next-generation sequencing techniques (RNA-seq, ChIP-seq).

11. "Methods of RNA structural biology".
Methods of studying the structures of RNA-protein complexes, methods of crystallization of RNP complexes (on the example of the ribosome, snRNP, RNase H). Comparison of crystallography and NMR studies for RNA. SAXS technique. Chemical (SHAPE) and bioinformatics prediction of RNA structures. Electron microscopy technique at low temperatures (Cryo-EM).

12. "Global analyzes of ribonucleoprotein complexes" part 1.
Biochemical methods of ribonucleoprotein (RNP) complexes purification. RNA chromatography and high-throughput RNP methods combined with NGS RNA-sequencing and mass spectrometry. Transcriptome, translatome and proteome analyses. High-throughput studies of chromatin, RNA structure and RNA-RNA, RNA-DNA and RNA-protein interactions. Methods of visualizing single RNA molecules, transcription and translation in living cells. Analysis of RNP complexes in cells by proximity labeling techniques.

12. "Global analyzes of ribonucleoprotein complexes" part 2.

Part of the lectures will be conducted by invited guests.

Practical part of the course (classes/lab work):

1. Basics of working with RNA. RNA isolation from yeast and plants.

2. RNA quality assessment. RNA electrophoresis. Radioisotope and fluorescent methods of RNA detection. miRNA detection in plants - introduction. miRNA detection in plants, Northern-blot technique (1) with RNA separation in polyacrylamide gel.

3. Northern-blot technique (1): hybridization with a biotin-labeled oligonucleotide probe. Washing, scanning and analysis of results. Detection of CUT transcripts in yeast: Northern-blot technique (2) with RNA separation in agarose gel.

4. Detection of CUT in yeast: radioactive probe labeling by "asymmetric PCR" and hybridization. Analysis of scientific images.

5. Detection of CUT in yeast: discussion of Northern-blot hybridization results (2). Analysis of the 3' ends of small nucleolar RNAs (snoRNAs) by cRT-PCR. Digestion of RNA-oligo nucleotide duplexes with RNase H and ligation (circularization) of RNA.

6. Analysis of the 3' ends of the snoRNA: RT + PCR.

7. cRT-PCR: separation of products in the gel and analysis of results. mRNA detection by RT-qPCR. Designing primers for qPCR - theory and practice. “Competition” for the most efficient pair of starters, i.e. each student will design at least 1 pair of starters, which will be ordered and tested during the next class.

8. Performing the qPCR reaction: testing the designed primers for chosen genes.

9. Analysis of qPCR results.

10. Biochemical determinations of the activity of RNA degrading enzymes. Analysis of the ribonucleolytic activities of different versions of the Dis3 protein - the main catalytic subunit of the exosome complex; testing the sensitivity of the 5'-3' exoribonucleolytic activity of the Xrn1 protein to the phosphorylation status of the 5' end of the substrate; degradation reactions of fluorescently labeled synthetic RNA oligonucleotides, separation of reaction products in a denaturing polyacrylamide gel.

11. Exploratory analysis of transcriptomic and translatomic data from RNA-seq experiments - bioinformatics exercises.

12. EMSA. Interactions of proteins and nucleic acids. Binding of yeast Nsi1 protein to rDNA. Fluorescent "gel-shift" (incubation of samples, separation in native polyacrylamide gel, scan).

13. Students’ presentations to pass the practical part of the course.