Catalytic RNAs, riboswitches and nucleoprotein complexes

1. Catalytic RNAs, riboswitches and nucleoprotein complexes The role of RNA in the cellular environment is more complex than originally thought. This is thought to be due to the facts that RNA can adopt complex 3D structures RNA can bind small molecule ligands with high affinity and specificity and this can lead to conformation changes RNA active sites can catalyse biochemical transformations

2. RNAs role as a ribozyme (RNA enzymes) Ribozymes are antisense RNA molecules that have catalytic activity • Enzymes made of protein are the dominant form of biocatalyst in modern cells • There are at least eight natural RNA enzymes, or ribozymes, that catalyze fundamental biological processes. It is believe that these ribozymes might be the remnants of an ancient form of life that was guided entirely by RNA. • Discovered by Thomas Czech, he got the Noble prize for it in 1989. The first ribozymes were discovered in Tetrahymena The structure of RNA indicates the potential to be catalytic. • It has the flexibility to fold into a three-dimensional, globular-like structure which can bring potential catalytic groups into close contact with the "substrate". • Both the phosphate backbone, the 2'-OH and the base have potential acid/base/nucleophile catalytic groups, • and metal ions bound to the phosphate can be electrophilic. RNA often controls the expression of genes, a role that had been thought to be at least mostly the domain of proteins called "repressors" and "transcription factors."

3. A number of labs around the world are now using these ribozymes to study gene function e.g. in the study of HIV, the AIDS virus, and in Cancer research. Mang Yu and coworkers at the University of California recently used ribozymes to provide white blood cells with resistance to HIV infection. A laboratory in Strasbourg is currently making use of ribozymes to study the involvement of certain gene sequences in the onset of Spinal Muscular Atrophy They designed ribozymes that target the RNA messenger of certain genetic sequences in muscle cells that have been cultured under special conditions and observed for cellular changes similar to those in diseased cells. Hopefully this will not only lead to a larger understanding of exactly what role such genetic sequences play in the onset of the disease but may also allow new potential therapies to be developed.

5. Examples of Ribozymes in bacterial cells and viruses • Plant viruses • Circular ribozymes, called viroids, have been discovered which can have a devastating effect on plants. • The ribozymes replicate themselves in copies attached to their own genome. • The viroids then undergo self-cleavage, sending fragments off to colonize other areas of the plant. • The viroids harm the plants by rapidly proliferating and using nucleotide materials the plant itself needs. • Further damage is caused as the viroid bundles interfere with the plant’s internal structures much like a tumor. • They have no protein producing capabilities. • Their cleavage from the mother strand is completely self-controlled and initiated. • They are catalytic in their own replication and processing. • A site is less than 30 nucleotides long and consists of three stems coming off a central loop is critical. • This secondary structure, called a "hammerhead", is capable of cleaving very specific sequences of RNA in order to release viable daughter strands of RNA. • Synthetic hammerheads, consisting of only 19 nucleotides, have already been produced, which can act as highly specific catalysts. • Similar synthetic ribozymes are being designed to break up RNA viruses and RNA involved in the transcription and translation of mutant DNA.

6. Ribozyme examples cont. 2. ribonuclease P (RNase P)- function to cleave a piece of RNA off a tRNA molecule RNase P is able to selectively cut more than 60 tRNA precursors, which then become mature tRNA molecules capable of carrying amino acids during the translation of proteins. .RNase P catalyzes specific phosphodiester bond hydrolysis in pre-tRNAs to produce mature tRNA 5'-ends. Without RNase P this process would not be possible. The enzyme is a ribonucleoprotein, although the RNA segment of the molecule has been shown to independently recognize and cleave the appropriate substrate both in vivo and in vitro. The protein segment of the RNase P appears to allow the ribozymal segment to work at a faster hydrolytic rate and with less Mg2+ present.

7. Ribozyme examples continued 3. The ribosome the atomic-level picture of the ribosome shows the complex fold of the RNA molecules buttressed and supported by numerous proteins. the peptidyltransferase center is composed of RNA, with no proteins in the vicinity This enzymatic activity is due to the RNA

8. . The active site of the ribosome consists of RNA (white strands), not protein (orange)

9. Riboswitches • The switching on and switching off of genes in response to an organism's needs is one of the most basic of biological control mechanisms. • Recently RNA elements built into messenger RNAs have been found to directly sense the concentration of small metabolites and turn gene expression on or off in response. • These riboswitches fold into intricate structures that can distinguish one metabolite from another . • Three distinct tricks for switching gene expression have been revealed: • the RNA element can cause premature termination of transcription of the mRNA, • it can block ribosomes from translating the mRNA, • or it can even cleave the mRNA and thereby promote its destruction. This involves an RNA unit directly binding a small-molecule metabolite, which switches the RNA into a conformation that activates its intrinsic self-cleavage activity. This "ribozyme riboswitch" represents a new type of biological activity for a catalytic RNA.

10. A riboswitch (A) binds to its target molecule (B) and inhibits nearby transcription machinery (C).

11. Riboswitches continued. • Aptamers are RNA or DNA molecules selected in vitro from vast populations of random sequence that recognize specific ligands by forming binding pockets. • Allosteric ribozymes are RNA enzymes whose activity is modulated by the binding of an effector molecule to an aptamer domain, which is located apart from the active site. • These RNAs act as precision molecular switches that are controlled by the presence or absence of a specific effector.

12. Riboswitches continued • Many antibiotics bind to ribosomal RNAs and selectively inhibit bacterial growth. • Riboswitches might also be targeted by new classes of antibiotics. • Given the significant role that riboswitches play in bacterial genetic control and the fact that they have evolved to bind metabolites, drug compounds could be created that disrupt bacterial genetic control. • Engineered riboswitches might function as designer genetic control elements. Natural Riboswitch Targets include: • Coenzyme B12 • Thiamine pyrophosphate • FMN • S-adenosylmethionine • Guanine • Adenine • Lysine

13. RNA Interference- another role for RNA • double-stranded RNA (dsRNA) is a potent regulator of gene expression . • Cells maintain a multi-step pathway for dealing with dsRNAs, either endogenous (those made by transcription of their own genes) or exogenous. • An enzyme called "dicer" cuts the dsRNAs into 20-base pair fragments. • One of the two strands is then transferred to a matching sequence on a messenger RNA, and an enzyme called "slicer" then cleaves the mRNA at the position of the duplex. • The cleaved mRNA is rapidly degraded. • In other cellular systems, instead of the mRNA being degraded it stays intact, but the presence of the short RNA duplex renders it somehow untranslatable, so no protein product is made. • The discovery of RNA interference (RNAi) has led to the identification of many small cellular RNAs that do not encode proteins but instead act to regulate the expression of other genes . • These microRNAs form extensively base-paired "foldback" structures that are then processed by RNAi. • RNAi has become a powerful tool for understanding which genes are important for which biological events.