RNA uses the sugar ribose instead of deoxyribose in its backbone

RNA uses the sugar ribose instead of deoxyribose inits backbone

Vicinal Hydroxyl Group Makes Difference

• The vicinal OH groups of RNA are susceptable to nucleophilic attach leading to hydrolysis of the phosphodiester bond.

• RNA is relatively resistant to the effects of dilute acid, but gentle treatment of DNA with 1mM HCl leads to hydrolysis of purine glycosidic bonds.

• DNA is not susceptible to alkaline hydrolysis. RNA is alkali labile.

RNA tends to be single-stranded

• Functional differences between RNA and DNA– DNA single function, – RNA many functions and forms

shallow minor groove

narrow/deep major groove

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RNA Structure & Function

• RNA structure• Levels of organization• Bonds & energetics

(more about this on Wed)

• RNA types & functions• Genomic information storage/transfer• Structural • Catalytic• Regulatory

D Dobbs ISU - BCB 444/544X: RNA Structure & Function

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Rob KnightUniv Colorado

RNA structure: 3 levels of organization


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Fig 6.2Baxevanis & Ouellette 2005

Covalent & non-covalent bonds in RNA

Primary: Covalent bonds

Secondary/Tertiary Non-covalent bonds

• H-bonds (base-pairing)

• Base stacking

Stem-loops are common elements of RNA structure.Stems are double-stranded regions of RNA that are A-form helices.(DNA is typically a B form helix)

Stem

Loop

The paired regions generally have an A – form right - handed helix.

Common secondary structure of RNAs. Bulge, internal loop, and haripin loop,

puesdoknots


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G-C, A-U, G-U ("wobble") & variants

Base-pairing in RNA

http://www.imbjena.de/ImgLibDoc/nana/IMAGE_NANA.html#sec_element

See: IMB Image Library of Biological Molecules

http://www.imbjena.de/ImgLibDoc/nana/IMAGE_NANA.html

http://www.imbjena.de/ImgLibDoc/nana/IMAGE_NANA.html

http://www.imb-jena.de/ImgLibDoc/nana/IMAGE_NANA.html

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Structures of mRNA

• Closely related to functions• E.g Riboswitches – part of mRNA that is

involved in gene regulation:– Metabolites bind molecules folded structure

recognizes metabolites– Conformation change results that switches off

gene expression

Recap

• RNA is structurally and functionally more versatile than DNA.

• RNA chains fold into unique three-dimensional structures which act similarly to globular proteins.

• Structure is the basis for their chemical reactivity and specific interactions with other molecules, including:– proteins, – nucleic acids – small ligands

Secondary Structures

• Based on conventional Watson and Crick base pairing but also unconventional pairing.

• Secondary structures of RNA can be predicted with good accuracy by computer analysis, based on thermodynamic data for the free energies of various conformations, comparative sequence analysis, and solved crystal structures.

Base-paired RNA adopts an A-type double helix

• In RNA, helix formation occurs by hydrogen bonding between base pairs and base stacking hydrophobic interactions within one single-stranded chain of nucleotides.

• X-ray crystallography studies have shown that base-paired RNA primarily adopts a right-handed A-type double helix with 11 bp per turn.

• Regular A-type RNA helices with Watson–Crick base pairs have a deep, narrow major groove that is not well suited for specific interactions with ligands.

• The minor groove does not display sequence specificity, it includes the ribose 2 -OH groups which are good hydrogen bond acceptors, ′and it is shallow and broad, making it accessible to ligands.

• Sites for RNA-binding proteins found commonly in the minor groove.

shallow minor groovedifficult to recognize

narrow/deep major groove

Tertiary structure of RNA

Additional Motifs of Tertiary Structure

• A minor motif• Pseudoknots• Tetraloops• Ribose zipper• Kink turn motif

Riboswitch

Secondary structure representation of the ligand-induced folding of the adenine-sensing riboswitch ligand-binding domain, as revealed by real-time 2D NMR.

Lee M et al. PNAS 2010;107:9192-9197

©2010 by National Academy of Sciences

Ninety eight percent of the human genome

does not code for protein. What is its

function?

How much of human transcribed RNAresults in proteins?

• Of all RNA, transcribed in higher eukaryotes, 98% are never translated into proteins.

• Of those 98%, about 50-70% are introns • 4% of total RNA is made of coding RNA• The rest originate from non-protein genes, including rRNA,

tRNA and a vast number of other non-coding RNAs (ncRNAs)

• Even introns have been shown to contain ncRNAs, for example snoRNAs

• It is thought that there might be order of 10,000 different ncRNAs in mammalian genome

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RNA functions

• Storage/transfer of genetic information

• Structural

• Catalytic

• Regulatory


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RNA functions

Storage/transfer of genetic information

• Genomes • many viruses have RNA genomes

single-stranded (ssRNA)e.g., retroviruses (HIV)

double-stranded (dsRNA)

• Transfer of genetic information • mRNA = "coding RNA" - encodes proteins


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RNA functions

Structural • e.g., rRNA, which is major structural component of

ribosomes BUT - its role is not just structural, also:

Catalytic RNA in ribosome has peptidyltransferase activity

• Enzymatic activity responsible for peptide bond formation between amino acids in growing peptide chain

• Also, many small RNAs are enzymes "ribozymes”

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RNA functions

Regulatory Recently discovered important new roles for RNAs In normal cells:

• in "defense" - esp. in plants• in normal development

e.g., siRNAs, miRNAAs tools:

• for gene therapy or to modify gene expression• RNAi (used by many at ISU: Diane

Bassham,Thomas Baum, Jeff Essner, Kristen Johansen, Jo Anne Powell-Coffman, Roger Wise, etc.)

• RNA aptamers (Marit Nilsen-Hamilton, ISU)

D Dobbs ISU - BCB 444/544X: RNA Structure & Function 46

RNA types & functions Types of RNAs Primary Function(s)

mRNA - messenger translation (protein synthesis) regulatory

rRNA - ribosomal translation (protein synthesis) <catalytic>

t-RNA - transfer translation (protein synthesis)

hnRNA - heterogeneous nuclear

precursors & intermediates of mature mRNAs & other RNAs

scRNA - small cytoplasmic signal recognition particle (SRP)tRNA processing <catalytic>

snRNA - small nuclear snoRNA - small nucleolar

mRNA processing, poly A addition <catalytic>rRNA processing/maturation/methylation

regulatory RNAs (siRNA, miRNA, etc.)

regulation of transcription and translation, other??

RNARNA

mRNAmRNA ncRNA(non-coding RNA) Transcribed RNA with a structural,

functional or catalytic role

ncRNA(non-coding RNA) Transcribed RNA with a structural,

functional or catalytic role

rRNARibosomal

RNAParticipate inprotein synthesis

rRNARibosomal

RNAParticipate inprotein synthesis

tRNATransfer RNA

Interface betweenmRNA &

amino acids

tRNATransfer RNA

Interface betweenmRNA &

amino acids

snRNASmall

nuclear RNA-Incl. RNA that

form part of the

spliceosome

snRNASmall

nuclear RNA-Incl. RNA that

form part of the

spliceosome

snoRNASmall

nucleolar RNAFound in

nucleolus,involved in

modificationof rRNA

snoRNASmall

nucleolar RNAFound in

nucleolus,involved in

modificationof rRNA

miRNAMicro RNA

Small RNA involved

regulation of expression

miRNAMicro RNA

Small RNA involved

regulation of expression

OthersIncluding large

RNAwith roles inchromotin

structure andimprinting

OthersIncluding large

RNAwith roles inchromotin

structure andimprinting

stRNASmall temporal RNA.

RNA with a role inDevelopmental timing.

stRNASmall temporal RNA.

RNA with a role inDevelopmental timing.

siRNASmall interfering RNAActive molecules in

RNA interference

siRNASmall interfering RNAActive molecules in

RNA interference

Small Nuclear RNAs

• One important subcategory of small regulatory RNAs consists of the molecules know n as small nuclear RNAs (snRNAs).

• These molecules play a critical role in gene regulation by w ay of RNA splicing.

• snRNAs are found in the nucleus and are typically tightly bound to proteins in complexes called snRNPs (small nuclear ribonucleoproteins, sometimes pronounced "snurps").

• The most abundant of these molecules are the U1, U2, U5, and U4/U6 particles, w hich are involved in splicing pre-mRNA to give rise to mature mRNA

MicroRNAs• RNAs that are approximately 22 to 26 nucleotides in length. • The existence of miRNAs and their functions in gene regulation w ere

initially discovered in the nematode C. Elegans .• Have also been found in many other species, including flies, mice, and

humans. Several hundred miRNAs have been identified thus far, and many more may exist.

• miRNAs have been show n to inhibit gene expression by repressing translation.

• For example, the miRNAs encoded by C. elegans, lin-4 and let-7, bind to the 3' untranslated region of their target mRNAs, preventing functional proteins from being produced during certain stages of larval development.

• Additional studies indicate that miRNAs also play significant roles in cancer and other diseases. For example, the species miR-155 is enriched in B cells derived from Burkitt's lymphoma, and its sequence also correlates w ith a know n chromosomal translocation (exchange of DNA between chromosomes).

Small Interfering RNAs

• Although these molecules are only 21 to 25 base pairs in length, they also work to inhibit gene expression.

• siRNAs were first defined by their participation in RNA interference (RNAi). They may have evolved as a defense mechanism against double-stranded RNA viruses.

• siRNAs are derived from longer transcripts in a process similar to that by which miRNAs are derived, and processing of both types of RNA involves the same enzyme, Dicer .

• The two classes appear to be distinguished by their mechanisms of repression, but exceptions have been found in which siRNAs exhibit behavior more typical of miRNAs, and vice versa.


C. elegans lin-4 Small Regulatory RNA

We now know that there are hundreds of microRNA genes

(Ambros, Bartel, Carrington, Ruvkun, Tuschl, others)

lin-4 precursor

lin-4 RNA

“Translational repression”

V. Ambros lablin-4 RNA

target mRNA

C Burge 2005


MicroRNA Biogenesis

N. Kim Nature Rev Mol Cell Biol 2005

C Burge 2005


miRNA and RNAi pathways

RISC

Dicerprecursor

miRNA siRNAs

Dicer

“translational repression”and/or mRNA degradation

mRNA cleavage, degradation

RNAi pathwaymicroRNA pathway

MicroRNA primary transcript Exogenous dsRNA, transposon, etc.

target mRNA

Drosha

RISCRISC

C Burge 2005


miRNA Challenges for Computational Biology

• Find the genes encoding microRNAs

• Predict their regulatory targets

• Integrate miRNAs into gene regulatory pathways & networks

Computational Prediction of MicroRNA Genes & Targets

C Burge 2005

Need to modify traditional paradigm of "transcriptional control" by protein-DNA interactions to include miRNA regulatory mechanisms

Documents

RNA uses the sugar ribose instead of deoxyribose in its backbone