66369 - Advanced Molecular Biology with Laboratory

Learning outcomes

With few exceptions, RNA has for a long time been merely regarded as a molecule that can either function as messenger (mRNA), or as part of the translation machinery (tRNA, rRNA). Recent findings in small RNA biology have demonstrated that these versatile molecules do not only play key roles in many important biological processess like splicing, editing, etc, but also can act catalytically, illuminating a staggering wealth of novel molecular mechanisms which regulate gene expression at the post-transcriptional level, in all kingdoms of life.
In eukaryotes, RNA interference (RNAi) has become a standard experimental tool and its therapeutic potential is being aggressively harnessed. Understanding the structure and function of small RNAs, such as siRNA and miRNAs, that trigger RNAi and down-regulate translation, has highlighted the assembly and function of the RNA-induced silencing complex RISC, providing new basic mechanisms of regulation, as well as guidelines to efficiently silence genes for biological research and therapeutic applications.
In bacteria, small RNAs are potent and multifunctional regulators, allowing new signalling pathways to cross-regulate targets independently of the transcriptional signals, introducing polarity within operons, modulating virulence, and explaining some puzzles in well studied regulatory-circuits.
These findings have profoundly changed our perception about how gene expression is regulated. This course aims to address the molecular biology of small regulatory RNAs, providing students with fundaments and cutting edge notions underlying one of the major paradigm shifts of modern biology.

Course contents

Introduction
Small, non-coding regulatory RNAs: a major paradigm shift in gene regulation. History, biological significance, implications, perspectives.
Fundaments
Analysis and discussion of seminal research articles describing the occurrence and the mechanisms underlying gene silencing mediated by small non-coding RNAs.
Insights
- Molecular mechanisms involved in small RNA processing and recognition: Drosha, Pasha, nuclear export, DICER, Argonautes, RISC assembly and function, strand recognition, comparison between siRNA and miRNA pathways, etc;
- Mechanisms of protein synthesis repression by miRNAs;
- P-bodies: mRNA purgatory;
- Silencing amplification in plants and C. elegans
- RNAi in the formation of heterochromatin;
- PIWIs & piRNAs: transposon silencing in the germline genome;
- Mirtrons
- endogenous siRNAs (esiRNA)
- Small regulatory sRNAs and CRISPRs in bacteria: ebiological function, molecular mechanisms, etc;

Readings/Bibliography

There is no need to purchase specific textbooks. Teaching material and articles will be made available through the AMS Campus - AlmaDL online repository.

Research Articles

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.
 
Reviews & Perspectives

General Review
Ghildiyal and Zamore (2009). Small silencing RNAs: an expanding universe. Nature Reviews Genetics. 10, 94-108.
 
Structure and Function of small regulatory RNAs
Rana (2007). Illuminating the silence: understanding the structure and function of small RNAs. Nature Reviews Mol Cell Biol. 8, 23-36.
 
miRNAs
Filipowicz et al. (2008). Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nature Reviews Genetics 9, 102-114.
Flynt and Lai (2008). Biological principles of microRNA-mediated regulation: shared themes amid diversity. Nature Reviews Genetics 9, 831-842.
 
piRNA
Aravin et al. (2007). The Piwi-piRNA Pathways Provides an Adaptive Defence in the Transposon Arms Race. Science 318, 761-764.
 
RITS and silencing of heterochromatin
Grewal and Elgin (2007). Transcription and RNA interference in the formation of heterochromatin. Nature 447, 399-406.
 
P-bodies
Eulalio et al. (2007). P bodies: at the crossroads of post-transcriptional pathways. Nature Reviews Mol. Cell. Biol. 8, 9-22.
Tritschler et al. (2010). Role of GW182 proteins and PABPC1 in the miRNA pathway: a sense of déjà vu. Nature Reviews Mol. Cell. Biol. 11, 379-384.
 
Argonautes
Hutvagner and Simard (2008). Argonaute proteins: key players in RNA silencing. Nature Reviews Mol. Cell. Biol. 9, 22-32.
Faehnle and Joshua-Tor (2010). Argonaute MID domain takes centre stage. EMBO reports 11, 564-565.
 
miRNA stability
Kai and Pasquinelli (2010). MicroRNA assassins:: factors that regulate the disappearance of miRNAs. Nature Structural and Molecular Biology 17, 5-10.
Rigoutsus and Furnari (2010). Decoy for microRNAs. Nature 456, 1016-1017.
 
esiRNA
Sasidharan and Gerstein (2008). Protein fossils live on as RNA. Nature 453, 729-731.

bacterial sRNAs
Waters and  Storz (2009). Regulatory RNAs in Bacteria. Cell 136, 615-628.
Horvath and Barrangou (2010). CRISPR/Cas, the Immune System of Bacteria and Archaea Sciene 327, 167-170.
Sontheimer and Marraffini (2010). Slicer for DNA Nature 486, 45-46.
Wagner (2009). Kill the messenger: bacterial antisense RNA promotes RNA decay. Nature Struct. Mol. Biol., 16, 804-806.
Belasco (2010). All things must pass: contrasts and commonalities in eukaryotic and bacterial mRNA decay. Nature Reviews Mol. Cell. Biol. 11, 467-478.

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

Assessment methods

Oral exam; lab protocol. The exam aims at the assessment of the expected learning outcomes. The final grade arises form the average scores of two oral exams,  comprising each three questions on the subjects taught. 

Teaching tools

.pdf files of seminal papers and scientific articles.
Powerpoint presentations of experimental approaches, results, and models.
Podcast reviews and insights (.mp4a).
Wetlab course

Registered students can download lesson presentations, articles and other teaching materials through te AMS campus ALma-DL site: http://campus.unibo.it/
using their login credentials.

Office hours

See the website of Alberto Danielli

See the website of Katia Scotlandi