72569 - Molecular Buology of Eukaryotes with laboratory

Course Unit Page

Academic Year 2018/2019

Learning outcomes

At the end of the course the student will master a method of study that will allow him to understand biological problems. 
The method will be 'applied to 2 different topics: 1) small non-coding RNAs involved in the process called "RNA interference" or RNAi and 2) proteomics and post-translational modifications of proteins with special insights on the modifications with ubiquitin or other similar peptides.
In particular, the student will learn how to:
- Analyze and discuss topics on regulation of gene expression by small RNAs;
- Propose strategies of inquiry into the biological function of miRNAs;
- Understand and critically analyze the literature;
- Design experiments on different Biomolecular problems.

Course contents

1. Model organisms for the study of gene function. C. elegans and the phenomenon of RNA interference (RNAi).
RNAi: gene interference by double-stranded RNA (dsRNA) in C. elegans. dsRNA directs the digestion of mRNA at intervals of 21-23 nucleotides. RNAi is mediated by RNA of 21-22 nucleotides.
Role of Dicer in RNAi. The genes and mechanisms regulating the expression of RNAi in C. elegans. Cloning and characterization of miRNAs. Asymmetry of the RISC complex. Molecular basis for the recognition of pri-miRNA by the complex Drosha/DGCR8.
The family of small RNA silencers: miRNA, siRNA and piRNA. Mechanisms of piRNA biogenesis.
Mechanisms of post-transcriptional regulation by miRNAs.
Transcription and RNAi in heterochromatin formation.

2. The complexity of the proteome. Post-translational modifications of proteins. Techniques for the study of proteins: antibodies, two-dimensional electrophoresis, protein identification by mass spectrometry. Experimental techniques for the study of protein-protein interactions: the yeast two-hybrid system in, GST-pull down, co-immunoprecipitation, purification of TAP-TAG.

3. The discovery of ubiquitin (Ub). The ubiquitination of proteins: the enzymatic cascade. Functional significance of mono- and poly-Ubiquitination. UBD proteins that bind Ub.
The family of SUMO proteins. The enzymatic cascade of sumoylation. Consensus sumoylation of canonical and non-canonical. Functional significance of sumoylation and possible models of operation.
Sumoylation assay in vivo and in vitro.

Readings/Bibliography

No text book is required. The course is based on scientific articles and other material that is sent to students by email.


Fire et al. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811.
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.
Schwarz et al. (2003). Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199-208.
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.
Han et al (2006). Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell 125, 887-901.
Buhler et al (2006). Tethering RITS to a nascent transcript initiates RNAi- and heterochromatin-dependent gene silencing. Cell 125, 873-886.
Ghildiyal and Zamore (2009). Small silencing RNAs: an expanding universe. Nature Reviews Genetics. 10, 94-108.
Rana (2007). Illuminating the silence: understanding the structure and function of small RNAs. Nature Reviews Mol Cell Biol. 8, 23-36.
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.
Aravin et al. (2007). The Piwi-piRNA Pathways Provides an Adaptive Defence in the Transposon Arms Race. Science 318, 761-764.
Grewal and Elgin (2007). Transcription and RNA interference in the formation of heterochromatin. Nature 447, 399-406.
Hershko A, Ciechanover A, Varshavsky A. Basic Medical Research Award. The ubiquitin system. Nat Med. 2000 Oct;6(10):1073-81.

Weissman AM. Themes and variations on ubiquitylation. Nat Rev Mol Cell Biol. 2001 Mar;2(3):169-78. Review.

Pickart CM, Cohen RE. Proteasomes and their kin: proteases in the machine age. Nat Rev Mol Cell Biol. 2004 Mar;5(3):177-87. Review.

Winget JM, Mayor T. The diversity of ubiquitin recognition: hot spots and varied specificity. Mol Cell. 2010 Jun 11;38(5):627-35. Review.Hicke L. Protein regulation by monoubiquitin. Nat Rev Mol Cell Biol. 2001 Mar;2(3):195-201. Review.Geiss-Friedlander R, Melchior F. Concepts in sumoylation: a decade on. Nat Rev Mol Cell Biol. 2007 Dec;8(12):947-56. Review.

Teaching methods

The course accredits a total of 9 credits. 7 cfu, equivalent to 56 hours, will be used for classroom lectures on the analysis and discussion of scientific articles and other supplementary material. 2 credits, equivalent to 30 hours, will be devoted to laboratory activities.

Assessment methods

The final grade is assigned to each student on the basis of a written test consisting of 6 individual groups of 6 questions regarding the topics (scientific papers) dealt with in class.
The written test is designed to assess the achievement of the following learning objectives:
Recognize and describe the experimental procedures used in the scientific articles discussed.
Understand and interpret the experimental results.

Teaching tools

Original scientific articles.
Copies of PowerPoint presentations used in the classroom
Laboratory of Molecular Biology.

Office hours

See the website of Davide Carlo Ambrosetti