Alan WeinerBIOCHEM 530Friday, EE 037October 10, 2014RNA structure and function

If RNA can be bothcatalyst and genome,

RNA could replicate itself… the first “living” molecule!

catalytic(+) strand

template(–) strand

catalytic (+) strand copies unfolded template (–) strand

two catalytic (+) strandsand original template (–) strand

Note that addition of a pppN to the 3' OH of a growing

RNA chain is nearly identical, chemically, to attack of a 3'

OH on the 5' or 3' splice site!

How the RNP got its protein…an offer it could not refuse?

independent RNAprotein makes an offer the RNA cannot refuse

RNA relaxes, now protein-dependent

Bruce Alberts (1986) The function of the hereditary materials: Biological catalyses reflect the cell's evolutionary history. Am Zoologist 26, 781-796

ribosome

spliceosomereverse transcriptase

metabolism

Catalytic RNA could have replicated itself before the advent of protein

synthesis!

Invention of the ribosome createda ribonucleoprotein (RNP) worldcomposed of RNA + proteins.

Invention of the spliceosome createdcomplicated proteins by exon shuffling.

Invention of reverse transcriptase began the transition from RNA genomes to DNA genomes, with retroviral-like elements as transitional genomes that persist today.

The RNP World is alive and well in our cells, but we are distracted by the vast genetic storage capacity of DNA!

excess iron sequestered by ferritin

scarce iron imported by transferrin receptor

Rouault and Klausner (1996) TiBS 21, 174; Beutler (2004) Science 306, 2051; Du et al. (2008) Science 320, 1088.

Translational control is powerful,and many proteins have "second jobs"

5’ UTR

3’ UTR

RNA is more than a structure or informational sequence…key steps in the "Central Dogma" are catalyzed and regulated by RNAs

DNA mRNA protein

mRNA splicingcatalyzed by small nuclear RNAs(snRNAs)

translation of mRNAs catalyzed byribosomal RNAs(rRNAs) and tRNAs

mRNA transcriptionregulated by long noncoding RNAs

(lncRNAs)chromatin structure regulated by small interfering RNAs(siRNAs and piRNAs)

defensive RNAs biosynthetic and regulatory RNAs

mRNA translation regulated

by microRNAs (miRNAs)and competing endogenousRNAs (ceRNAs)

The amazing world of RNA interference (RNAi)

miRNAs, siRNAs, ceRNAsand their relatives

Ghildiyal and Zamore (2009) Small silencing RNAs:an expanding universe. Nat Rev Genet 10, 94-108; Broderick and Zamore (2011) MicroRNA therapeutics. Gene Therapy 18, 1104-1110.

RNA interference (RNAi)

Fighting fire with fireor

How Nature uses RNAs

to regulate themselves,

chromatin structure, and transcription

2006 Nobel Laureates Andrew Fire (center), Craig Mello (right),

and Alfred Nobel (left, unsmiling)

Caenorhabditis elegans(aka C. elegans, "the worm", nematode, roundworm)

1 mm

Zamore and Haley (2005) Ribo-gnome: The big world of small RNAs. Science 309, 1519-1524; Ghildiyal and Zamore (2009) Small silencing RNAs: an expanding universe. Nat Rev Genet 10, 94-108

The human genome is chock full of two dispersed, highly repeated families of retroelements called L1's or "LINEs" (long interspersed repeated elements) and Alu's or "SINEs" (short interspersed repeated elements). L1 and Alu elements both contain promoters, and RNA transcripts of these elements are reverse transcribed into DNA copies which insert into new, almost random sites throughout our genome. L1 elements encode a reverse transcriptase and DNA integrase that enables them to move to new sites, whereas Alu elements — which have no protein coding capacity — must borrow the L1 reverse transcriptase and integrase in order to retrotranspose.

The big question: Why aren't our genomes overun or destroyed by this infestation of mobile, parasitic DNA elements? Use RNA to fight RNA!

Alu RNA

Alu RNA

genomic DNA Alu geneL1 L1

L1 RNA

L1 RNA

Alu Alu

Alu RNA

The human genome is chock full of parasitic retrotransposable elements (“retrotransposons” aka “retroposons”) that use a

reverse transcriptase to replicate and transpose through RNA intermediates

LINEs

SINEs

autonomous

non-autonomous

autonomous

non-autonomous

850,000

1,500,000

21%

13%

endogenousretrovirus-like

elements450,000 8%

6-8 kb

}6-11 kb

1.5-3 kb

100-300 bp

AAAORF1 RT-INT

AAA

gag RT-INT env

gag

DNA transposonfossils

autonomous

non-autonomous300,000 3%

2-3 kb

80-3,000 bp}transposase

( )

length

copy numb

er

percentage

of genome

structure of retroelemen

tclass

autonomy

integration

retroviral particle

extracellular

intracellular

RNA

RNA

retroviral particle

RNA

cDNA

DNA

cDNA

RNA

reverse transcriptio

n

second strand synthesis

transcription

provirusgenome

translation to generate reverse transcriptase (RT), integrase, and capsid proteins for viral

assembly

integration

RNA

RNA

cDNA

DNA

cDNA

RNA

reverse transcriptio

n

second strand synthesis

transcription

parental retroelementgenome

translation of LINE RNA to generate reverse transcriptase

and integrase also used by noncoding SINEs

daughter retroelement

+RT

RNA

+RT

LINEs and SINEs may move from cell to cell, organism to organism,and species to species,as stowaways in viral

particles.

stowaway RNAs

Retroviruses and retrotransposable elements made (ridiculously) simple

Retroposition of SINEs and LINEs requires portable promoter and termination signals within the RNA transcription unit: this is possible because the transcription initiation factors that position RNA polymerase at the initiation site can do their job from upstream or downstream, and the polyadenylation signal is transcribed and preserved within the mature RNA transcript.

RNAPTIF

+1

RNAP TIF

+1

“conventional” externalpromoter

internal“portable”promoter

pA

pA

AAAAA

AAAAA

Surprisingly few diseases are caused by spontaneous retroposition of SINEs and LINEs

Although 1,500,000 SINE insertions and 850,000 LINE insertions account for >34% of the complete human genome sequence, SINEs are responsible for only 25 or 0.05% of all known disease-causing mutations, and LINEs for only 5 or 0.01% of the total.

Callinan and Batzer (2006) Retrotransposable elements and human disease. Genome Dyn 1, 104; Plasterk (2002) RNA Silencing: The Genome's Immune System. Science 296, 1263

Most of these 1,500,000 SINE insertions and 850,000 LINE insertions have degenerated in place by neutral mutation and are no longer mobile. The few mobile SINEs and LINEs that have caused spontaneous disease are “young,” i.e. they have inserted recently and have not yet had time to degenerate.THE BIG QUESTION: What protects our genomes from being blasted to bits by retroposition of SINEs and LINEs? The answer appears to be that RNAi is our DNA genome’s immune system!

Six easy steps to understanding RNAi

Step One. A perfect RNA duplex is usually a sign of danger.

RNA

RNA

viral DNA genome

bidirectional transcription of a compact viral genome

Alu RNA

Alu RNA

genomic DNA

transcription of dispersed repeated retroelements ("genomic parasites")

Alu geneL1 L1

L1 RNA

Alu and L1 RNA

Alu Alu

Six easy steps to understanding RNAi

Step Two. The RNA duplex is "diced" into 21 bp fragments.

RNA

RNA

"dicer"-likeenzymes

21 bp siRNAs

long RNAs

Six easy steps to understanding RNAi

Step Three. One RNA strand associates with a RISC-like complex

(RISC = RNA-Induced Silencing Complex)

RNA

RNA

"dicer"-likeenzymes

21 bp duplex siRNAs

long duplex RNAs

one RNA strand associates with a RISC-like protein

complex

21 base siRNAsin active

complexes

RISC RISC RISC RISC RISC

5’

RNApolymerase

DNA

nascent RNA

nucleus

cytoplasm

5’ AAAA 3’

mature mRNA

RISC

RISC-like

nucleosomes mRNA attenuation,

degradation, or even activation

by miRNAs

chromatin silencingby siRNAs and

piRNAs

Step Four. A RISC-like complex anneals with complementary RNA, regulating or degrading the RNA and/or silencing the corresponding

chromatin.

Six easy steps to understanding RNAi

Step Five. Our genome encodes >1200 regulatory microRNAs.

mRNA

cap AAAAAORF

imperfectmatch

translationalattenuation

(usually)

unprocessed microRNA (miRNA)

transcript

1. miRNA excisionby "dicer"-like

enzymes

2. assembly of the miRNAinto a RISC-like complex

RISCDrosha

Dicer

Guo et al. (2010) Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466, 835-840; Friedman et al (2009) Most mammalian mRNAs are conserved targets of microRNAs. Genome Res 19, 92-105; Bartel (2009) MicroRNAs: target recognition and regulatory functions. Cell 136, 215-233.

Six easy steps to understanding RNAi

Step Six. Match with mRNA determines degradation or attenuation.

mRNA

cap AAAAAORF

RISC mRNA

cap AAAAAORF

imperfectmatch

attenuation

perfectmatch

degradation

RISC

Six easy steps to understanding RNAi…

Step One. A perfect RNA duplex is usually a sign of danger.

RNA

RNA

viral DNA genome

bidirectional transcription of a compact viral genome

Alu RNA

Alu RNA

genomic DNA

transcription of dispersed repeated retroelements ("genomic parasites")

Alu geneL1 L1

L1 RNA

Alu Alu

Alu and L1 RNA

5’

RNApolymerase

DNA

nascent RNA

nucleus

cytoplasm

5’ AAAA 3’

mature mRNA

RISC

RISC-like

nucleosomes mRNA attenuation,

degradation, or even activation

by miRNAs

Step Four. A RISC-like complex anneals with complementary RNA, regulating or degrading the RNA and/or silencing the corresponding

chromatin.

chromatin silencingby siRNAs and

piRNAs

de novo silencing maintenance of silencing

transcription

dsRNA formation

RNAi-mediated processing

recruitment of histone methyltransferase

local histone H3K9 methylation

recruitment of chromodomain proteins

Dernburg and Karpen (2002) Cell 111, 159-162; Coulmenares et al. (2007) Mol Cell 27, 449-461

RNAi amplification by RNA-dependent RNA

polymerase

(worms not humans)

DNA replication,proteins dissociate,

histones rebind locally

proteins rebind to methylated histones

methylation spreads, DNA resilenced

chromodomain protein

histone methyltransferase

methylatedhistone H3

KEY

a

b

c

d

Wang et al. (2008) Nature 456, 209-213

Thermus thermophilus argonaute bound to a 5'-phosphorylated 21-base DNA guide strand

The DNA (red) is bound in a deep channel where the sugar-phosphate backbone is held ina helical conformation and the Watson/Crick bases are stacked and ready for base pairing...thus Argonaute proteins are Nature's own way of making "locked nucleic acids" (LNAs).

()

Here is Herpesvirus saimiri HSUR1 titrating out host miRNAs miR-142-3p and miR-27a

Cazalla et al. (2011) A Primate Herpesvirus Uses the Integrator Complex to Generate Viral MicroRNAs. Mol Cell 43, 982.

So, naturalists observe, a fleaHas smaller fleas that on him prey;And these have smaller still to bite‘em;And so proceed ad infinitum.

Jonathan Swift (1667–1745)Poetry, A Rhapsody

coding ceRNA(competing

endogenous RNA)

regulated mRNA

noncoding ceRNAs(aka lncRNA for long noncoding RNA) may bind miRNAs to linear sequences or multiple

proteins to 3D structures

3’ UTRmiRNA

cap AAAAAA

cap AAAAAA

cap AAAAAAORF

ORF3’ UTR

cap AAAAAA

Molecular circuitry by which lnc-MD1, miR-135, and miR-133 regulate muscle

differentiation

Cesana et al. (2011) A long noncoding RNA controls muscle differentiation by functioning as a competing endogenous RNA. Cell

147, 358-369

lnc-MD1

AnAn

microRNAs repress muscle mRNAs through 3' UTR lnc-MD1 RNA relieves

repression by soaking up miRNAs

lnc-MD1

So, naturalists observe, a fleaHas smaller fleas that on him prey;And these have smaller still to bite‘em;And so proceed ad infinitum.

Jonathan Swift (1667–1745)Poetry, A Rhapsody

coding ceRNA(competing

endogenous RNA)

regulated mRNA

noncoding ceRNAs(aka lncRNA for long noncoding RNA) may bind miRNAs to linear sequences or multiple

proteins to 3D structures

3’ UTRmiRNA

cap AAAAAA

cap AAAAAA

cap AAAAAAORF

ORF3’ UTR

cap AAAAAA

A model for lncRNA (long noncoding RNA) function in cellular circuitry. (left) Embryonic Stem (ES) cell-specific transcription factors (such as Oct4, Sox2 and Nanog) bind to the lncRNA promoter and drive transcription. The lncRNA binds to ubiquitous regulatory proteins, giving rise to cell-type specific RNA-protein complexes. Through different combinations of protein interactions, the lncRNA-protein complex gives rise to unique transcriptional programs. (right) A similar process may work in other cell types with specific transcription factors regulating lncRNAs, which form cell type-specific RNA-protein complexes that regulate cell-type-specific gene expression programs.

lncRNAs act in the circuitry controlling pluripotency and

differentiation [Guttman et al. (2011) Nature 477, 295]

If RNA is everywhere, and does everything, why is it such a challenging

drug target?

aspirin, 180 Da

ibuprofen, 206 Da

The monomer unit of RNA is a 330 Da ribonucleotide; an RNA polymer of 15 monomer units is called an oligonucleotide (or “15-mer”) and would be 5,000 Da

CH3

Why RNA doesn't look "druggable" at first glance

PPPPPP PPP PPPP

PPPPPP PPP PPPP

unmodifiedRNA or DNA

membranebilayer

polyanions cannot penetrate the negatively charged membrane bilayer

Why unmodified oligonucleotides are not good drugs

X

P–P

–P–P

–P–P

–P

P–P

–P–P

–P–P

–P

– –– –– –– –– –– –

PP

PP

PP

PP

PP

cellulartarget RNA

antisenseoligonucleotide

P–P–P–P–P–P–P

cap AAAAAmRNA ORF

The antisense specificity problem is huge: there is no escape from "off-target" effects

cap AAAAAmRNA ORF

cap AAAAAmRNA ORF

cap AAAAAmRNA ORF

cap AAAAAmRNA ORF

cap AAAAAmRNA ORF

oligo too short, binds too weakly

oligo too long, bindsto unintended targets("off-target effects")

"magic bullet" may not exist, biologyis too complicated!

H

H

DNA with phosphorothioate linkages

RNA with phosphorothioate linkages

OH

OH

RNA with phosphorothioate linkages and 2'-O-methyl

modifications

OCH3

O

CH3

siRNAs that cannot be digested by exonucleases and endonucleases, and/or cannot cleave themselves

What is a peptide-nucleic acid or PNA? The neutral peptide backbone in peptide-nucleic acid (PNA) replaces the negatively charged sugar-phosphate backbone of RNA and DNA.

The resulting PNA:RNA or PNA:DNA duplex is much more stable than a pure RNA or DNA duplex, because there is no charge/charge repulsion between the two strands.

PNA is also resistant to degradation by cellular nucleases and proteases.

proteinDNA(or RNA)PNA

peptide bond

Koppelhus and Nielsen (2003) Adv Drug Deliv Rev 55, 267-280;Ivanova et al. (2008) Nucl Acids Res 3, 6418-6428.

LNAs ("locked nucleic acids") enable shorter RNAsto achieve specificity and resist cellular nucleasesA locked nucleic acid (LNA) is an RNA

monomer in which a bridge connecting the 2' oxygen and 4' carbon locks the ribose sugar into the conformation it would normally adopt in an RNA double helix. This conformation enhances base stacking, backbone pre-organization, and increases the melting temperature of the duplex; however, it also prevents LNAs from functioning as a ribozymes. LNA oligomers can be synthesized chemically.

In a therapeutic proof-of-principle, cardiac function and survival both improved when LNA oligomers complementary to miR-208a were injected into the tail vein of hypertensive rats [Montgomery et al. (2011) Circulation 124, 1537].

baseC

locked nucleic acid (LNA)

baseC5'

3'

H2'

4'

RNA monomerin double helix