RNAi: Insight, Mechanisms and Potential Nobel Prize in Physiology or Medicine awarded to A. Fire and C. Mello

What is RNA interference (RNAi)? “The Process by which dsRNA silences gene expression...” Degradation of mRNA or translation inhibition

What are sense and antisense RNA? Messenger RNA (mRNA) is single- stranded, called "sense" because it results in a gene product (protein). 5´ C U U C A 3´ mRNA 3´ G A A G U 5´ Antisense RNA

What are sense and antisense RNA? Antisense molecules interact with complementary strands of nucleic acids, modifying expression of genes. 5´ C U U C A 3´ mRNA 3´ G A A G U 5´ Antisense RNA

RNAi terms dsRNA: double stranded RNA, longer than 30 nt miRNA: microRNA, nt. –Encoded by endogenous genes siRNA: small-interfering RNA, nt. –Mostly exogenous origin

Nature, it seems, is the popular game for milliards, and milliards, and milliards of particles playing their infinite game of billiards and billiards and billiards Piet Hein (1966) * Piet Hein (December 16, April 17, 1996), a Danish scientist, mathematician, inventor, author, and poet; known for his short poems in the form of grook. A grook ("gruk“, short for "GRin & sUK" ("laugh & sigh" in Danish) is a form of short aphoristic poem. *From ‘Critical Mass: How One Thing Leads To Another’ by Phillip Ball

RNAi like phenomena Plants –Petunias Fungi –Neurospora Animals –Caenorhabditis elegans

Alternate terms to RNAi PTGS (Posttranscriptional Gene Silencing) CosuppressionQuelling Virus-induced gene silencing

1990-Petunias Napoli et al. defined an RNAi-like phenomenon and called it “cosupression.” chalcone synthase (CHS), a key enzyme in flavonoid biosynthesis, the rate-limiting enzyme in anthocyanin biosynthesis, responsible for the purple coloration.

Overexpression of chalcone synthase in petunias unexpectedly resulted in white petunias The levels of endogenous as well as introduced CHS were 50-fold lower than in wild- type petunias, which led the authors to hypothesize that the introduced transgene was “cosuppressing” the endogenous CHS gene.

1992-The mold Carlo Cogoni and Guiseppe Macino of the Università di Roma La Sapienza in Italy introduced a gene needed for carotenoid synthesis in the mold Neurospora crassa: –The introduced gene led to inactivation of the mold's own gene in about 30% of the transformed cells. They called this gene inactivation "quelling." A rosette of the asci

1995-The worm Guo and Kemphues studied par-1 gene during embryogenesis The worm, C. elegans –has a fixed lineage: hypodermis, intestine, gonads –asymmetric divisions

1995- The worm Guo and Kemphues first studied Par-1 gene mutants –Division: Asymmetric  symmetric –P-granule distribution

Guo and Kemphues, 1995

Both the antisense and sense strands effectively silenced wildtype Par-1 RNAi

The reasonings about the wonderful and intricate operations of Nature are so full of uncertainty, that, as the Wise-man truly observes, hardly do we guess aright at the things that are upon earth, and with labour do we find the things that are before us. Stephen Hales (1727) Stephen Hales (September 17, January 4, 1761) was an English physiologist, chemist and inventor; studied the role of air and water in the maintenance of both plant and animal life.

‘Antisense’ Technology? Sense RNA silences yet no hybridization of sense RNA with sense mRNA is expected! Intronic and promoter sequences do not silence. ssDNA or dsDNA does not work! Craig Mello at the Worm Meeting in Madison, Wisconsin coined the term ‘RNAi’ and said that: –“ We can’t call it ‘antisense’ when ‘sense’ works as well”* *Montgomery (2006) RNA interference: unraveling a mystery

Craig Mello In 1996, C. Mello and his student S. Driver also reported that sense RNAs mimic antisense phenotype. –Injection is made into a single site yet acts more systemically.

Andrew Fire In 1991, A. Fire successfully targeted genes by antisense constructs from transgenes. Sense constructs also exhibited silencing activity.

Fire and Mello Their paths crossed: –Mutual interest on a gene called pie-1. –Discuss what the sense and antisense preparations have in common

Fire and Mello did simple math… Sense + (-sense)  0 (interference)

Fire & Mello hypothesize on how sense works? ssRNA in vitro synthesized by bacteriophage RNA pol might be contaminated by dsRNA Bacteriophage RNA polymerase produce random ectopic transcripts

DNA transgene arrays produce aberrant RNA some with dsRNA character certain repeated DNA sequences might be expected to produce aberrant RNAs that would then be capable of producing a silencing response Fire & Mello hypothesize on how sense works?

1998-Fire et al. and Mello, Nature

1998-Fire et al and Mello Gel-purified ssRNA Used purified ssRNA (antisense and sense) separately and also together. Tested ssRNA against different genes for specificity Tested whether a general post- transcriptional silencing is in place.

Unc-22 (Uncoordinated 22) Codes for a non essential myofilament It is present several thousand copies/cell

[If] There is some precision, there is some science. Herbert Spencer (1880) Herbert Spencer (27 April 1820 – 8 December 1903) was an English philosopher and prominent classic-liberal political theorist. Spencer analyzed human societies as evolving systems, and coined the term "survival of the fittest."

Injection for RNAi 6-10 adult hermaphrodites were injected with 0.5x x10 6 molecules into each gonadal arm.

Unc-22 phenotype 4-6 hours after injection, eggs collected. Screened for phenotypic changes –twiching ExonSizeRNAPhenotype Exon SenseAntisenseSense+antisenseWildtypeWildtype Twicher (100%) Exon SenseAntisenseSense+antisenseWildtypeWildtype Twicher (100%)

Mex-3 mex-3 encodes two RNA binding proteins; in the early embryo, maternally provided Mex-3 is required for specifying the identities of the anterior AB blastomere and its descendants, as well as for the identity of the P3 blastomere and proper segregation of the germline P granules

Mex-3 RNAi b, Embryo from uninjected parent (showing normal pattern of endogenous mex-3 RNA20). c, Embryo from a parent injected with purified mex-3B antisense RNA. Retain the mex-3 mRNA, although levels may be somewhat less than wild type. d, Embryo from a parent injected with dsRNA corresponding to mex- 3B; no mex-3 RNA is detected.

RNAi concentration and dose response 3.6x10 6 molecules/gonad –Sense phenocopied 1% of progeny –Antisense phenocopied 11% of progeny –dsRNA phenocopies 100% progeny and at even 3x10 8 molecules/gonad.

Quantitative Assays

Other possibilities Sense+antisense in low salt Rapid sequential injection of sense & antisense –Both cause interference –1 hour apart injection of sense and antisense leads to reduction in interference.

Conclusions

Conclusions

Nobody should be rich but those who understand it Wolfgang von Goethe Johann Wolfgang Goethe (28 August 1749 – 22 March 1832), a German polymath: a poet, novelist, dramatist, humanist, scientist, theorist, painter; the author of Faust and Theory of Colours

Nobel Prize in Physiology or Medicine 2006 Andrew Fire Craig Mello

The Laureates

Essense of Nobel Prize Mello’s words after he had heard the news: “I seem too young, and isn’t the gap unusually short?” Most Nobel prizes are given many years after the relevant discovery. The Fire and Mello award, given just eight years after publication of their paper, is reminiscent of Kary Mullis’s 1993 chemistry Nobel. That prize was awarded for his 1985 invention of the polymerase chain reaction — a method of gene amplification that invaded research labs just as fast and comprehensively as the RNAi technique has. Nature, News. Allison Abbott

RNAi studies in PubMed Years before 2007 Number of Publications

Ways to induce silent phenotypes Timmons and Fire showed that feeding dsRNA works! Reversible and gene-specific effects…

Ways to induce silent phenotypes Tabarra, Grishok, and Mello in 1998 demonstrated that soaking in dsRNA also works! Nomarski image showing embryos produced by a wild-type mother treated with pos-1 RNAi by soaking. All except one embryo (arrow) show the distinctive pos-1 embryonic arrest with no gut, no body morphogenesis, and extra hypodermal cells pos-1 encodes a CCCH-type zinc-finger protein; maternally provided POS-1 is essential for proper fate specification;

Mechanisms revealed 25bp species of dsRNA found in plants with co- suppression [Hamilton and Baulcombe, 1999] Sequence similar to gene being suppressed Drosophila: long dsRNA “triggers” processed into 21-25bp fragments [Elbashir et al., 2001] –Fragments = short interfering RNA (siRNA) –siRNA necessary for degradation of target

RNAi: two phases Initiation –Generation of mature siRNA or miRNA Execution –Silencing of target gene –Degradation or inhibition of translation

RNAi illustrated RNAi illustrated

How does RNAi work?

siRNA biogenesis Dicer (type III RNAse III) cleaves long dsRNA into siRNA 21-25nt dsRNA from exogenous sources –Symmetric 2nt 3’ overhangs, 5’ phosphate groups –Evidence for amplification in C. elegans and plants

RNA Induced Silencing Complex (RISC) RNAi effector complex Preferentially incorporates one strand of unwound RNA [Khvorova et al., 2003] –Antisense How does it know which is which? –The strand with less 5’ stability usually incorporated into RISC [Schwarz et al., 2003]

siRNA design Mittal, 2004

Custom-made siRNAs

siRNA libraries Generation of a feeding clone Tuschl, 2003

siRNA libraries Result: bacterial strains 86.3% of predicted genes with RNAi phenotypes assigned

Assayed Phenotypes: Examples Emb – embryonic lethal Ste – sterile Gro – slow growth Adl – adult lethal Lvl – larval lethality Lva – larval arrest Bmd – body morphological defects Unc – uncoordinated Clr – clear Prz – paralyzed Lon – long Mlt – moulting defects Egl – egg laying defects Him – high incidence of males

Endogenous RNAi-miRNA We have hundreds of different genes that encode small RNA (collectively, microRNA) whose precursors can form double-stranded RNA. These can activate the RNA interference process and thus switch off the activity of various genes with matching segments. First miRNA is lin-4

More miRNAs No other miRNAs found for 7 years! –Second miRNA – let-7 [Reinhart et al., 2000] –Non coding, 21nt RNA –Regulates lin-14 in same way as lin-4 Years before 2007 Number of Publications

Defense Against Viruses Indeed, Baulcombe, Vance, and others have shown that, in the continuing evolutionary war to survive and reproduce, plant viruses have evolved genes that enable them to suppress silencing.

Mammalian RNAi McManus and Sharp, 2002

Getting Around the Problem siRNA (21-22nt) mediate mammalian RNAi –Introducing siRNA instead of dsRNA prevents non-specific effects

Some applications of RNAi Therapy –Candidate genes, drug discovery, and therapy Genome-wide RNAi screens –Gene function –Candidate genes and drug discovery Systems biology –Models of molecular machines

Drugging the worms AGING, CANCER, NEURODEGENERATION, NEUROBIOLOGY

Genome-wide Screens

Early Embryonic Phenotypes Osmotic integrity Passage through meiosis Entry into interphase Cortical dynamics Nuclear appearance Sister chromatid separation Asymmetry of division Cytokinesis Pace of development Centrosome attachment

Genome-wide RNAi A total of 19,075 genes were targeted by dsRNAs. More than 40,000 time-lapse microscopy recordings, scored and annotated.

Genome-wide RNAi Only 11% genes showed detectable RNAi phenotype Between genes are required for early embryogenesis.

Systems Biology and RNAi Cellular systems act as networks of interacting components (genes, RNA, protein, metabolites,…). Genome-wide RNAi screens offers the potential for revealing functions of each protein. Combining RNAi screen data with other highthroughput data (e.g., protein-protein interaction, mRNA expression profiling) leads to understanding of the organization of the cell system.

‘Tis true, There’s magic in the web of it. William Shakespeare (1602-4) The Future: Integrative Biology

Networks of Early Embryogenesis Protein-protein interaction dataset: binary physical interactions between 3,848 C. elegans proteins Transcriptome dataset: expression profiling similarity above a given threshold among genes in the network Phenotypic dataset: phenotypic similarity above another threshold of 661 early embryogenesis genes. RNA interference (RNAi) phenotypic signature consisting of a vector describing specific cellular defects in early embryogenesis.

Systems Biology Approach: Three networks in one

Does it make sense to combine?

The embryogenesis network

The aesthetics of natural science and mathematics is at one with the aesthetics of music and paintings both inhere in the discovery of a partially concealed pattern. Herbert Simon (1996) Herbert Alexander Simon (June 15, 1916 – February 9, 2001), an American political scientist and polymath; one of founding fathers of Artificial Intelligence, information processing, decision-making, and complex systems, the first to analyze the architecture of complexity and to propose a preferential attachment mechanism to explain power law distributions.

1995-Guo & Kemphues

A book: Critical Mass by Phillip Ball How one thing leads to other He details the development of key concepts in contemporary physics, such as self-organization, and chaos; Next, he shows how social scientists apply these concepts to the study of human organization. the relationship between global phenomena and local actions.

The reader will appreciate the orderliness of the lines… and he will see how this orderliness points to the existence of a fundamental governing principle. George Kingley Ziph (1949)

Links to endogenous dsRNA

1999-Hamilton & Baulcombe Hypothesis: Antisense RNA complementary to the target mRNA may be too short to observe so is easy to miss. Tomato lines transformed with ACO cDNA under the 35S promoter.

1999-Hamilton & Baulcombe

1999-Hamilton & Baulcombe concluded:

Discovery Project

C. Elegans as a Disease Model AGING, NEURODEGENERATION, NEUROBIOLOGY –Longevity model: daf-2 mutants and RNAi showed that reduced insuling signaling increases resistance ox

Discovery of miRNA Discovery of the first miRNA, lin-4 –Non-coding, 22nt RNA –Identified in screen for defects in timing of larval development lin-4 mutation – ectopic larval stage 1-like cell divisions at later stages lin-14 mutations – reciprocal phenotype, same regulatory pathway as lin-4 –lin-4 negatively regulates lin-14 translation –lin-4 partially complementary to conserved sites in lin-14 3’UTR [Lee et al., 1993] –Required for negative regulation of lin-14 –lin-4 binds these sites

More miRNAs miRNA is in other organisms? –Let-7 Homologs were easily detected [Pasquinelli et al., 2000] Drosophila, sea urchins, mice, humans... Indicates RNAi general conserved mechanism

RNAi in other genes Hlh-1: myogenic regulatory factor (MRF) subgroup of bHLH proteins; HLH-1 activity is required during embryonic development for the proper differentiation and function of body wall muscle cells

RNAi in other genes Unc-54: encodes a muscle myosin class II heavy chain (MHC B) required for locomotion and egg-laying

Defense against transposons RNAi may also help keep the transposable elements that litter genomes from jumping around and causing harmful mutations. Plasterk's team and Mello, Fire, and their colleagues found that mutations that knocked out RNAi in C. elegans led to abnormal transposon movements.

I have endeavored to show that it is the peculiar function of physical science to lead us to the confines of the comprehensible, and to bid us behold it and receive it in faith, till such time as the mystery shall open. James Clerk Maxwell (1856) Rethinking of the antisense dogma When 19th century Scottish physicist James Clerk Maxwell was faced with predicting the behavior of gases made up of trillions of rapidly moving molecules, he borrowed from social scientists the mathematical tools of statistics and successfully applied them to the problem.