27534 - Advanced Molecular Biology 1

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 inhibit 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 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 RNAs in bacteria: early days, modern times, biological function, molecular mechanisms, Fur, RhyB, Hfq, etc;
- aRNAs, etc.

Readings/Bibliography

Fundaments

Articles
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 Nucleotide 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
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
Schwarz et al. (2003). Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199-208.
Song et al (2004). Cristal structure of Argonaute and its implications for RISC silencer activities. Science 305, 1434-1437
Buhler et al (2006). Tethering RITS to a nascent transcript initiates RNAi- and heterochromatin-dependent gene silencing. Cell 125, 873-886.


Reviews & Perspectives

Really New Advances. The Economist Jun 14th 2007. Traduzione Nuovi Paradigmi Internazionale 701, Jul 13th 2007, 48-51

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.
Aravin et al. (2007). The Piwi-piRNA Pathways Provides an Adaptive Defence in the Transposon Arms Race. Sceince 318, 761-764.
Grewal and Elgin (2007). Transcription and RNA interference in the formation of heterochromatin. Nature 447, 399-406.
Eulalio et al. (2007). P bodies: at the crossroads of post-transcriptional pathways. Nature Reviews Mol. Cell. Biol. 8, 9-22.
Hutvagner and Simard (2008). Argonaute proteins: key players in RNA silencing. Nature Reviews Mol. Cell. Biol. 9, 22-32.
Sasidharan and Gerstein (2008). Protein fossils live on as RNA. Nature 453, 729-731.
Buchan and Parker (2007). Two faces of miRNA. Science 318, 1877-1878.
Gottesman et al (2006). Small RNA regulators and the bacterial response to stress. . 71, 1-11.


Insights

miRNA
Lee et al (1993). The C. elegans Heterochronic Gene lin-4 Encodes Small RNAs with Antisense Complementarity to lin-14. Cell 75, 843-854
Reinhart et al (2000). The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403, 901-906
Tuschl/Bartel/Ambros Labs (2001). Identification of Novel Genes Coding for Small Expressed RNAs / An abundant class of Tiny RNAs with Probable Regulatory Roles in Caenorhabditis elegans / An Extensive Class of Small RNAs in Caenorhabditis elegans. Science 294, 853-864

Quelling
Cogoni et al. (1996). Transgene silencing of the al-1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic effector and does not depend on DNA-DNA interactions or DNA methylation. EMBO J. 15, 3153-3163.
Cogoni and Macino (1997). Isolation of quelling-defective (qde) mutants impaired in posttranscriptional transgene-induced gene silencing in Neurospora crassa. PNAS 94, 10233-10238.

Molecular mechanisms of miRNA processing and RISC maturation
Gregory et al (2004). The microprocessor complex mediates the genesis of of microRNAs. Nature 432, 235-240
Denli et al (2004). Processing of primary microRNAs by the microprocessor complex. Nature 432, 231-235
Liu et al (2004). Argonaute 2 is the catalytic engine of mammalian RNAi Science 305, 1437-1441
Okamura et al (2004). Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways. Genes Dev. 18, 1655-1666.
Lee et al (2004). Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell, 117, 69-81.
Yi et al. (2003). Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev. 17, 3011-3016.
Matranga et al (2005). Passenger-strand cleavage facilitate assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell 123, 607-620.
Rand et al. (2005). Argonaute 2 cleaves the anti-guide strand of siRNA during RISC activation. Cell 123, 621-629.


Mechanisms of inhibition of protein synthesis (miRNA)
Pillai et al (2005). Inhibition of translational initiation by let-7 microRNAs in human cells. Science 309, 1573-1576
Wu et al (2006). MicroRNAs direct radip deadenylation of mRNA. PNAS 103, 4034-4039.
Wakiyama et al (2007) let-7 microRNA-mediated mRNA deadenilation and translational repression in a mammalian cell-free system. Genes Dev 21, 1857-1862
Chendrimada et al (2007) MicroRNA silencing through RISC recruitment of eIF6. Nature 447, 823-829
Pillai et al. (2007). Repression of protein synthesis by miRNAs: how many mechanisms? TRENDS Cell. Biol. 17, 118-126.

P-bodies: mRNA purgatory
Liu et al. (2005). MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies. Nature Cell Biol. 7, 719-723
Sen and Blau (2005). Argonaute 2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies. Nature Cell Biol. 7, 633-636
Eulalio et al. (2007). P bodies: at the crossroads of post-transcriptional pathways. Nature Reviews Mol. Cell. Biol. 8, 9-22.
Parker and Sheth (2007). P Bodies and the Control of mRNA Translation and Degradation. Molecular Cell 25, 635-646.

Small RNAs & heterochromatin silencing
Volpe et al (2002). Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 297, 1833-1837.

endo-siRNAs
Czech et al (2008). Nature 453, 798-802
Ghildyal et al (2008). Science 320, 1077-1081
Kawamura et al (2008). Nature 453, 793-797
Okamura et al (2008). Nature 453, 803-806
Tam et al. (2008). Nature 453, 534-538
Watanabe et al. (2008). Nature 453, 539-543

PIWIs & piRNAs
O'Donnell and Boeke (2007). Mighty Piwis Defend the Germline against Genome Intruders. Cell 129, 37-44.
Brennecke et al. (2007). Discrete Small RNA-Generating Loci as Master Regulators of Transposon Activity in Drosophila. Cell 128, 1089-1103.

Mirtrons
Ruby et al. (2007). Intronic microRNA precursors that bypass Drosha processing. Nature 448, 83-86.
Okamura et al. (2007). The mirtron pathway generates microRNA-class regulatory RNAs in Drosophila. Cell 130, 89-100.

Silencing Amplification
Cogoni and Macino (1999). Gene silencing in Neurspora crassa requires a protein homologous to RNA-dependent RNA polymerase. Nature 399, 166-169.
Lipardi et al. (2001). RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell 107, 297-307.
Axtell et al. (2006). A two-hit trigger for siRNA biogenesis in plants. Cell 127, 565-77.
Pak and Fire (2007). Distinct populations of primary and secondary effectors during RNAi in C. elegans. Science 315, 241-244.
Sijen et al. (2007). Secondary siRNAs result from unprimed RNA synthesis and form a distinct class. Science 315, 244-247.
Baulcombe (2007). Amplified Silencing. Science 315, 199-200.


Bacterial small regulatory RNAs (sRNA)
Mizuno et al. (1984). A unique mechanism regulating gene expression: translational inhibition by a complementary RNA transcript (micRNA). PNAS 81, 1966-1970.
Altuvia et al. (1997). A small, stable RNA induced by oxidative stress: role as a pleiotropic regulator and antimutator. Cell 90, 43-53.
Massé and Gottesman (2002). A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli. PNAS 99, 4620-4625.
Massé et al (2003). Coupled degradation of a small regulatory RNA and its mRNA targets in Escherichia coli. Genes Dev. 17, 2374-2383.
Lenz et al (2004). The small RNA chaperone Hfq and multiple small RNAs control quorum sensing in Vibrio harveyi and Vibrio cholerae. Cell 118, 69-82.
Aiba (2007). Mechanism of RNA silencing by Hfq-binding small RNAs. Curr. Opin. Micriobiol. 10, 134-139.

aRNAs
Li et al (2006). Small dsRNAs induce transcriptional activation in human cells. PNAS 103, 17337-17342
Janowski et al (2007). Activating gene expression in mammalian cells with promoter-targeted duplex RNAs. Nature Chem. Biol. 3, 166-173
Erika Check (2007). RNA interference: Hitting the on switch. News Feature. Nature 448, 855-858
Vasudevan et al. (2007). Switching from Repression to Activation: miRNAs can upregulate translation. Science 318, 1931-1934.

Teaching methods

Introductory lessons to principal themes
Analysis and in class discussion of seminal research papers
Summarizing powerpoint presentations and podcasts

Assessment methods

Oral exam

Teaching tools

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

Office hours

See the website of Alberto Danielli