C8497 - RNA Biology and Riboregulation

Learning outcomes

Upon completion of the course, students will have acquired a thorough understanding of eukaryotic and prokaryotic mechanisms of RNA-mediated regulation, including RNAi, miRNA, piRNA, sRNA, ribonucleotides, and CRISPR. Students will be able to correctly understand the significance of new discoveries in these fields, evaluate their potential applications, and critically integrate this knowledge into a biotechnological context. Specifically, students will acquire expertise in gene regulation mechanisms mediated by noncoding RNAs, derived ablation strategies, and CRISPR-based methodologies.

Course contents

Riboregulation and RNAi

A major paradigm shift in biology. Quelling. Co-suppression in plant. Genetic bases of RNAi. Molecular dissection of RNAi, dicing and slicing. DICER. RISC assembly and maturation, guide strand selection, ARGONAUTES. endo-siRNA, pseudogenes.

microRNAs

Heterochronic gene regulation, miRNA biogenesis, DROSHA, microprocessor, mirtrons, miRNA trimming, nuclear export. mechanisms of miRNA-dependent regulation, mRNA decay, P-bodies. Network motifs and miRNA: a Systems Biology perspective.

ncRNAs and epigenetic regulation.

Silencing amplification, RdRPs, RNAi and heterochromatin, RITS, nuclearRNAi. piRNAs and PIWIs: guarding the germline, lncRNAs.

Bacterial regulatory ncRNAs

Riboswitches, sRNAs, 6S RNAs, CRISPRs

Genome editing methodologies

CRISPR applications, ZFN, TALENs, Cre-lox, Flp

WET-LAB COURSE

Functional analysis of the post-transcriptional regulation mediated by the RyhB sRNA with GFP reporter-fusions

Methods

• Site-specific mutagenesis
• Molecular cloning
• DNA miniprep
• Restriction analysis
• Plasmid incompatibility groups
• GFP reporter analysis
• Molecular microbiology techniques
• Multiplate reader measurements, fluorimetry

Readings/Bibliography

Teaching material and articles will be made available through the Insegnamenti Online (Moodle) repository.

Additional readings

Watson. Molecular Biology of the Gene. 7th edition. Chapter 7

Research Articles to be critically analyzed

RNAi discovery
Fire et al. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811.

21-23 nt dsRNA effectors; DICER
Zamore et al (2000). RNAi: Double-Stranded RNA Directs the ATP-Dependent Cleavage of mRNA at 21 to 23 Nucleoide Intervals. Cell 101, 25-33.
Berstein et al (2001). Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363-366.

RISC assembly and maturation
Schwarz et al. (2003). Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199-208.

microRNA regulation and RNAi share common genes and effector mechanisms
Grishok et al (2001). Genes and Mechanisms Related to RNA Interference Regulate Expression of the Small Temporal RNAs that Control C. elegans Developmental Timing. Cell 106, 23-34.

the MICROPROCESSOR complex
Han et al (2006). Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell 125, 887-901.

RITS and heterochromatin silencing
Buhler et al (2006). Tethering RITS to a nascent transcript initiates RNAi- and heterochromatin-dependent gene silencing. Cell 125, 873-886.

Bacterial non-coding regulatory RNAs
Pfeiffer et al (2009). Coding sequence targeting by MicC RNA reveals bacterial mRNA silencing downstream of translational initiation. Nature Struct. Mol. Biol., 16, 840-847.

Teaching methods

Introductory lessons to principal themes
Analysis and in class discussion of seminal research papers

Summarizing powerpoint presentations and podcasts
Lab: post-transcriptional regulation mediated by the RhyB sRNA in Escherichia coli

 

Wetlab course NOTE! The wetlab training activity requires that students take modules 1 and 2 in e-learning mode [https://www.unibo.it/it/servizi-e-opportunita/salute-e-assistenza/salute-e-sicurezza/sicurezza-e-salute-nei-luoghi-di-studio-e-tirocinio] and participate to module 3 of specific training on safety and health in the workplace. Information on dates and attendance of module 3 can be found in the appropriate section of the course website.

Assessment methods

Oral exam; lab protocol. The exam aims at the assessment of the expected learning outcomes. The final grade arises from three questions on the subjects taught.

SERVICE FOR STUDENTS WITH DISABILITIES AND SLD

Students with specific learning difficulties (SLD) or temporary or permanent disabilities: please contact the relevant University Office in good time (https://site.unibo.it/studenti-con-disabilita-e-dsa/it). The office will be responsible for proposing any necessary adjustments to the students concerned; these must, however, be submitted for approval by the lecturer at least 15 days in advance, and the lecturer will assess their suitability, taking into account the course’s learning objectives.

Students recognized as “working students”
Please consult the dedicated website (https://www.unibo.it/en/study/guide-to-choosing-your-programme/balancing-study-and-work) to apply for this status and to learn about the available measures.

Teaching tools

.pdf files of seminal papers and scientific articles.
Powerpoint presentations of experimental approaches, results, and models.

Wet lab activity

Registered students can download lesson presentations, articles and other teaching materials through the Insegnamenti Online site: https://virtuale.unibo.it
using their login credentials.

Office hours

See the website of Alberto Danielli

See the website of Paolo Emidio Costantini