RNA uses the sugar ribose instead of deoxyribose in its 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

14 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 15 Rob Knight Univ Colorado RNA structure: 3 levels of organization

D Dobbs ISU - BCB 444/544X: RNA Structure & Function 16 Fig 6.2 Baxevanis & 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

D Dobbs ISU - BCB 444/544X: RNA Structure & Function 23 G-C, A-U, G-U ("wobble") & variants Base-pairing in RNA See: IMB Image Library of Biological MoleculesIMB Image Library of Biological Molecules

24

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 groove difficult 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: ©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 RNA results 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

42 RNA functions Storage/transfer of genetic information Structural Catalytic Regulatory

D Dobbs ISU - BCB 444/544X: RNA Structure & Function 43 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

D Dobbs ISU - BCB 444/544X: RNA Structure & Function 44 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”

45 RNA functions Regulatory Recently discovered important new roles for RNAs In normal cells: in "defense" - esp. in plants in normal development e.g., siRNAs, miRNA As 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 & Function46 RNA types & functions Types of RNAsPrimary Function(s) mRNA - messengertranslation (protein synthesis) regulatory rRNA - ribosomaltranslation (protein synthesis) t-RNA - transfertranslation (protein synthesis) hnRNA - heterogeneous nuclearprecursors & intermediates of mature mRNAs & other RNAs scRNA - small cytoplasmicsignal recognition particle (SRP) tRNA processing snRNA - small nuclear snoRNA - small nucleolar mRNA processing, poly A addition rRNA processing/maturation/methylation regulatory RNAs (siRNA, miRNA, etc.) regulation of transcription and translation, other??

RNA mRNA ncRNA(n on-coding RNA) Transcribed RNA with a structural, functional or catalytic role ncRNA(n on-coding RNA) Transcribed RNA with a structural, functional or catalytic role rRNA Ribosomal RNA Participate in protein synthesis rRNA Ribosomal RNA Participate in protein synthesis tRNA Transfer RNA Interface between mRNA & amino acids tRNA Transfer RNA Interface between mRNA & amino acids snRNA Small nuclear RNA -Incl. RNA that form part of the spliceosome snRNA Small nuclear RNA -Incl. RNA that form part of the spliceosome snoRNA Small nucleolar RNA Found in nucleolus, involved in modification of rRNA snoRNA Small nucleolar RNA Found in nucleolus, involved in modification of rRNA miRNA Micro RNA Small RNA involved regulation of expression miRNA Micro RNA Small RNA involved regulation of expression Others Including large RNA with roles in chromotin structure and imprinting Others Including large RNA with roles in chromotin structure and imprinting stRNA Small temporal RNA. RNA with a role in Developmental timing. stRNA Small temporal RNA. RNA with a role in Developmental timing. siRNA Small interfering RNA Active molecules in RNA interference siRNA Small interfering RNA Active 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.

D Dobbs ISU - BCB 444/544X: RNA Structure & Function52 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 lab lin-4 RNA target mRNA C Burge 2005

D Dobbs ISU - BCB 444/544X: RNA Structure & Function53 MicroRNA Biogenesis N. Kim Nature Rev Mol Cell Biol 2005 C Burge 2005

D Dobbs ISU - BCB 444/544X: RNA Structure & Function54 miRNA and RNAi pathways RISC Dicer precursor miRNA siRNAs Dicer “translational repression” and/or mRNA degradation mRNA cleavage, degradation RNAi pathway microRNA pathway MicroRNA primary transcript Exogenous dsRNA, transposon, etc. target mRNA Drosha RISC C Burge 2005

D Dobbs ISU - BCB 444/544X: RNA Structure & Function55 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