Non-coding RNAs

1. Non-coding RNAs or RNAs come more than in three flavours...

2. How big part 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 • 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

3. The two main classes of ncRNAs • Housekeeping ncRNAs, which are constitutively expressed and required for normal function and viability of cell • Regulatory ncRNAs are expressed only in certain stages of organism development or as a response to external stimuli. • Regulatory ncRNAs can affect the expression of other genes at the level of transcription or translation

4. Housekeeping ncRNAs • tRNA and rRNA - translation • snRNA – Pre-mRNA splicing • snoRNA – rRNA modification • gRNA – guide RNA in RNA editing • Telomerase RNA – primer for telomeric DNA synhesis • A few other...

5. 4.5S RNA and 7S RNA – a part of signal recognition particle (SRP) Signal sequence • SRP recognizes signalling amino acid sequence in the N-terminus of growing polypeptide chain • Upon signal recognition, ribosome is attached to endoplasmatic reticlum so that the protein, made by ribosome, enters the secretory pathway ribosome SRP mRNA SRP receptor SRP translocon Endoplasmatic reticlum membrane

6. Structure of SRP RNA holds together the protein subunits of SRP as well as helps to bind to ribosome

7. tmRNA and trans-translation • tmRNA is a hybrid molecule, half tRNA, half mRNA • tmRNA helps to rescue ribosomes, bound to mRNA which lacks the termination codon • In addition, tmRNA adds a degradation signal to nascent protein

8. 3’ • If the termination codon by some mistake is not reached, the ribosome gets stack upon the reaching of 3’ end of mRNA and has to be rescued • Since the stop codon is not reached, the newly made protein is probably wrong and needs to be degraded

9. Ala • The tRNA part of tmRNA (black) adds an alanine to the growing polypeptide chain • The mRNA part (red) enters the ribosome and the synthesis of polypeptide is continued with aid of normal tRNAs (blue), until the termination codon is reached • In the end, ribosome is released and the newly made fusion protein is degraded due to the signal sequence in C-terminus 3’

10. Regulatory ncRNAs • Transcriptional regulators • Translational regulators • Modulators of protein function • Regulators of RNA and protein distribution

11. Dosage compensation • In animals, males and females have different number of X chromosomes (e.g. 1 or 2) • To equalize the expression levels from X chromosome in males and females some sort of mechanism must exist, called dosage compensation

12. Dosage compensation mechanisms

13. The role of roX ncRNAs in dosage compensation in Drosophila roX1 and roX2 ncRNAs are expressed only in males and they are responsible for for MSL (Male Specific Lethal) complex assembly. The MSL complex acetylates H4 histones on X chromosomes therefore increasing the transcription level MSL complex has about 35 entry sites in Drosophila genome. Two of them actually contain roX1/rox2 gene. This suggests a possible role of rox1/rox2 RNAs in entry site recognition MLE helicase MSL-2 MSL-1 MOF histone acetylase MSL-3 2 x MSL-3 roX1/roX2 ncRNAs

14. Acetylated lysines

15. Silencing of one female X chromosome in mammals • The X chromosome silencing is mediated by Xist – a 16,000 nt long ncRNA • Xist ncRNA recruited complex has one entry site in X chromosome, corresponding to Xist gene itself • Xist appears to recruit a specific histone isoform – H2A1.2 which maintains the chromosome in inactive state • Additionally, Xist containing complexes recruit histone deacetylases and methylases • Xist activity is regulated by another 40,000 nt long ncRNA – Tsix, which contains anti-sense sequence of Xist and therefore is able to regulate Xist activity by base-pairing to it

16. Genetic imprinting and shRNAs • Genetic imprinting is a process which results in expression on only one allele of gene, while the allele originating from the other parent is silenced • Process is somewhat similar to dosage compensation • The differences of expression from both alleles are due to different states of chromatin (euchromatin and heterochromatin) and also to differential methylation of DNA • Activity of small heterochromatic RNAs (shRNAs) appear to be essential for establishing and maintaining the imprinted status of genes • Activity of various shRNAs is not limited only to genetic imprinting

17. DNA and RNA recognition models of shRNA initiated chromatin condensation

18. Translational regulation • Translational regulation by ncRNAs is achieved by anti-sense mechanism, when ncRNA binds to target mRNA • RNA interference – covered separately in the end of this lecture

19. HFE promoter Sense exons Anti-sense promoter Anti-sense exons HFE mRNA A Ribosome B Anti-sense RNA Translation of human HFE gene is downregulated by anti-sense RNA

20. DsrA RNA in E.coli activates ribosome binding to stress-response s factor rpoS mRNA DsrA RNA Ribosome binding site blocked by base-pairing rpoS mRNA RBS accessible

21. Protein function modulation • Some ncRNAs can bind directly to proteins, altering their structure, enzymatic activities or ligand binding • Targets of such ncRNAs often are proteins, involved in transcription, for example nuclear receptors or general transcription factors

22. 6S RNA modulates s70 function in E.coli Log-phase Stationary phase s70 RNA pol + + + 6S RNA

23. Ribozymes • RNA molecules with catalytical properties (Ribonucleic acid enzymes) • In nature ribozymes occur mostly within self-splicing intrones and RNA encoded parasites – satellites and viroids • The catalyzed reactions in naturally occuring ribozymes are limited to cleavage and ligation of RNA • Some researchers consider even ribosomes being ribozymes, since the peptide bond formation is catalyzed by RNA • Most naturally occuring ribozymes act on themselves • The catalytical efficiency of ribozymes is typically much lower (~1000-fold) than of analogous protein enzymes • Several synthetic ribozymes are cabaple of performing other reactions than RNA cleavage and ligation

24. Cleaving ribozymes

25. The general secondary structure of hammerhead ribozyme Cleavage Y=C or T, R=A or G, H=A,T or C Dot represents any nucleotide

26. The 3D structure of hammerhead ribozyme

27. Hammerhead ribozyme mechanism Requires bivalent metal ion for activity

28. Other classes of cleaving ribozymes do not require metal ion for activity. Amino group of nearby nucleotide base destabilizes the phosphodiester bond instead Cyt

29. Metabolite-responsive ribozyme-mRNA hybride

30. Ligating ribozymes Reaction mechanism similar to that of RNA polymerases, requires Mg ion for catalysis

31. RNAse P RNAse P cleavage site

32. RNAse P is a ribozyme • RNAse P cleaves the 5’ end of pre-tRNAs • It is composed of 12 kDa P protein and about 400 nt long RNA • The catalytic activity lies entirely within RNA part • Enzyme is efficient without P protein but in high salt conditions • P protein or high salt is thought to screen the repulsive electrostatic interactions between RNAse P RNA and substrate pre-tRNA

33. One sequence – two ribozymes • Synthetic RNA molecule, capable to acquire 2 completely different secondary structures • Each structure performs different enzymatic activity: ligation versus cleavage • Based on two different initial ribozymes with similar length

34. RNA interference (RNAi) A natural biological mechanism for silencing genes Revolutionary new technology (potent and simple) to knock down gene expression in eukaryotic cells

35. 1998 1999 2000 2001 2002 2003 2004 RNA interference (RNAi) 2000 RNAi article amount 1500 1000 500 0 year

36. How was RNAi discovered? The injection of double-stranded RNAs into C. elegans resulted in the silencing of a genecomplementary to dsRNAs. A- negative control (without hybridization probe) B- normal pattern of endogenous mex-3 RNA C- injected with antisense RNA D- injected with dsRNA

37. So how does this silencing process work? RISC - RNA induced silencing complex siRNA – silencing RNA

38. Defense mechanism against dsRNA-containing viruses May stabilize the genome by sequestering repetitive sequences such as mobile genetic elements Control cellular development Dicer knockout mice don’t survive past gastrulation RNAi is widespread among eukaryotes Highly evolutionarily conserved property Must have important functions!

39. RNAi technology limitations in mammalian systems chemically synthesized siRNA dsRNA ( >30 nt ) cleaved by Dicer in vitro transcribed dsRNA general interferon response effective but transient silencing of gene expression global inhibition of mRNA translation

40. siRNA- mediated RNAi is transient RNAi in: Fungi, plants and worms Drosophila and mammals • cell – autonomous silencing • non – heritable • no indication of siRNA replication • systemic nature of silencing • heritable • can replicate siRNA with RNA-dependent RNA polymerases

41. RNAi versus miRNA translational repression • micro RNAs (miRNAs) are not perfectly complimentary to their targets • miRNAs do not induce target cleavage but block translation by binding to complementary mRNAs • miRNAs are encoded by the host genome, whereas siRNAs in most cases originate from outer source

42. Is RNAi exclusively limited to cytoplasm and post-transcriptional control ? • Although this is a very common view, it does not always have to be the case • siRNA can be transported to nucleus and act as shRNA to block transcription

43. DNA and RNA recognition models of shRNA initiated chromatin condensation

44. The RNA world – did it exist? • Probably, yes

45. The modern world The RNA world DNA RNA RNA Proteins information flow Information carryer replication

46. The main requirement of RNA world... • If there was an RNA world, there must have been an RNA molecule which is itself capable of making RNA, or in other words – an RNA ploymerase, made of RNA • So far, such a primordial polymerase is not known to exist in nature • However a synthetical RNA molecule, capable to replicate RNA has been made

47. Isolated from a pool of about 1015 synthetic RNAs, based on ligating ribozyme • Fidelity of 96.7 % • Extension time: 14 nucleotides in 24 hours

48. Late RNA world • 1) ribozymes, able to catalyze peptide bond formation and other chemical reactions emerged. Such ribozymes have been made in vitro. • 2) proteins began to take over the enzymatic activities

49. The pre-RNA world • The available synthetic ribopolymerase is 165 nt long. Even one tenth of that is far too long to emerge accidentally in the prebiotic soup • Some researchers argue that some sort of yet unknown simpler polymer must have existed before RNA