46678 - Advanced Molecular Biology I

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, 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
- Repression of the host immune response by viral ncRNAs
- Small regulatory RNAs in bacteria: early days, modern times, biological function, molecular mechanisms, Fur, RhyB, Hfq, etc;
- Provoking hypotheses and curiosities: 20K ORFs vs 30K miRNAs, aRNAs, RNAi screens, etc.

Readings/Bibliography

Fundaments

RNAi
Guo and Kemphues (1995). par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asimmetrically distributed. Cell 81, 611-620.
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

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.

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

Insights

Molcular mechanisms involved in recognition and processing

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

Export
Yi et al. (2003). Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev. 17, 3011-3016.

Dicers
Lee et al (2004). Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell, 117, 69-81.
Pham et al (2004). A Dicer-2-Dependent 80S Complex Cleaves Targeted mRNAs during RNAi in Drosophila. Cell 117, 83-94.
Tijsterman and Plasterk (2004). Dicers at RISC: The Mechanism of RNAi. Cell 117, 1-4.

Argonauti
Liu et al (2004). Argonaute 2 is the catalytic engine of mammalian RNAi Science 305, 1437-1441
Song et al (2004). Cristal structure of Argonaute and its implications for RISC silencer activities. Science 305, 1434-1437
Okamura et al (2004). Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways. Genes Dev. 18, 1655-1666.
Förstemann et al (2007). Drosophila microRNAs Are Sorted into Functionally Distinct Argonaute Complexes after Production by Dicer-1. Cell 130, 287-297.
Tomari et al (2007). Sorting of Drosophila Small Silencing RNAs. Cell 130, 299-308.

RISC
Schwarz et al. (2003). Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199-208.
Khvorova et al. (2003). Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209-216.
Haley and Zamore (2004). Kinetic analysis of the RNAi enzyme complex. Nature Struct. Mol. Biol. 11, 599 - 606.
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.
Gregory et al (2005). Human RISC Couples microRNA Biogenesis and Posttranscriptional Gene Silencing. Cell 123, 631-640.
Preall and Sontheimer (2005). RNAi: RISC Gets Loaded. Cell 543-545.
Ameres et al (2007). Molecular Basis for Target RNA Recognition and Cleavage by Human RISC. Cell 130, 101-112.

Rana (2007). Illuminating the silence: understanding the structure and function of small RNAs. Nature Reviews Mol Cell Biol. 8, 23-36.

Mechanisms of protein synthesis inhibition (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
Thermann and Hentze (2007). Drosophila miR2 induces pseudo-polysomes and inhibits translation initiation. Nature 447, 875-878.
Pillai et al. (2007). Repression of protein synthesis by miRNAs: how many mechanisms? TRENDS Cell. Biol. 17, 118-126.

P-bodies: mRNA purgatory
Sheth and Parker (2003). Decapping and Decay of Messenger RNAOccur in Cytoplasmic Processing Bodies. Science 300, 805-808.
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
Behm-Ansmant et al (2006). mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes. Genes Dev. 20, 1885-1898
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.

RNAi and heterochromatin silencing
Volpe et al (2002). Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 297, 1833-1837.
Zilberman et al (2003). ARGONAUTE4 Control of Locus-Specific siRNA Accumulation and DNA and Histone Methylation. Science 299, 716-719.
Pal-Bhadra et al (2004). Heterochromatic Silencing and HP1 Localization in Drosophila Are Dependent on the RNAi Machinery. Science 303, 669-672.
Verdel et al (2004). RNAi-Mediated Targeting of Heterochromatin by the RITS Complex. Science 303, 672-676.
Chicas et al. (2005). Small interfering RNAs that trigger posttranscriptional gene silencing are not required for the histone H3 Lys9 methylation necessary for transgenic tandem repeat stabilization in Neurospora crassa. Mol Cell Biol. 25, 3793-801.
Irvine et al (2006). Argonaute Silencing Is required for Heterochromatic Silencing and Spreading. Science 313, 1134-1137.
Grimaud et al (2006). RNAi Components Are Required for Nuclear Clustering of Polycomb Group Response Elements. Cell 124, 957-971.
Lei and Corces (2006). A Long-Distance Relationship between RNAi and Polycomb. Cell 124, 886-888.
Bernstein and Allis (2005). RNA meets chromatin. Genes Dev. 19, 1635-1655.
Grewal and Elgin (2007). Transcription and RNA interference in the formation of heterochromatin. Nature 447, 399-406.


PIWIs & piRNAs
O'Donnell and Boeke (2007). Mighty Piwis Defend the Germline against Genome Intruders. Cell 129, 37-44.
Lin (2007). piRNAs in the Germ Line. Science 316, 397.

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.
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.

Viral immunosuppressive miRNAs
Stern-Ginossar et al (2007). Host immune system gene targeting by a viral miRNA. Science 317, 376

Small regulatory RNAs in bacteria

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.
Zhang et al (1998). The OxyS regulatory RNA represses rpoS translation
and binds the Hfq (HF-I) protein
EMBO J. 17, 6061-6068.
Zhang et al (2002). The Sm-like Hfq protein increases OxyS RNA interaction with target mRNAs. Mol Cell 9, 11-22.
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.
Carpousis (2003) Degradation of targeted mRNAs in Escherichia coli: regulation by a small antisense RNA. Genes Dev. 17, 2351-2355.
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.
Afonyushkin et al (2005). Both RNase E and RNase III control the stability of sodB mRNA upon translational inhibition by the small regulatory RNA RyhB. Nucelic Acid Research 33, 1678-1689.
Morita et al (2005). RNAse E-based ribonucleoprotein complexes: mechanical basis of mRNA destabilization mediated by bacterial noncoding RNAs. Genes Dev. 19, 2176-2186.
Vecerek et al (2007). Control of Fur synthesis by the non-coding RNA RhyB and iron-responsive decoding. EMBO J. 26, 965-975
Gottesman (2005). Micros for microbes: non-coding regulatory RNAs in bacteria. Trends Genet. 21, 399-404.
Aiba (2007). Mechanism of RNA silencing by Hfq-binding small RNAs. Curr. Opin. Micriobiol. 10, 134-139.


Provoking hypotheses and curiosities

aRNA
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

etc…
Berezikov et al. (2006). Diversity of microRNAs in human and chimpanzee brain. Nature Genetics 38, 1375-1377.
The ENCODE Project Consortium (2007). Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 447, 799-816.
Greally (2007). Encyclopaedia of humble DNA. Nature 447, 782-783.

Teaching methods

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

Assessment methods

Admission Test (pass/fail).
Oral examination.

Teaching tools

Analysis and discussion of seminal papers and scientific articles.
Powerpoint presentations of experimental approaches, results, and models.
Podcast reviews of main themes.

Office hours

See the website of Alberto Danielli