transcription activators, repressors, & control RNA splicing, procesing and editing

transcription activators, repressors, & control RNA splicing, procesing and editing

1. RNA splicing, processing & editing Submitted by Ranjitha H B P-2082 BTY

2. Processing of eukaryotic pre-mRNA Conversion of primary transcript synthesized by RNA polymerase II to functional mRNA. Processing occur in nucleus as nascent mRNA is being transcribed 5’ capping 3’ cleavage/ polyadenylation RNA splicing RNA editing

3. 5̍ ́capping After nascent RNA, reach a length of 25-30 nucleotides 7-methylguanosine added through unusual 5’,5’- triphosphate linkage Capping enzyme associates with phosphorylated CTD of RNA polymerase II

4. Pre mRNA are associated with hnRNP proteins Pre- mRNA associates with heterogeneous ribonucleoprotein particles ( hnRNPs ) Modular structure- RNA binding domain & interacting domain RNA binding motifs- RRM-RNA recognition motif RGG box KH motif Functions - prevents formation of 2˚ structures - Makes pre mRNA substrate for processing - Transport of mRNA

5. 3’ cleavage and polyadenylation All mRNA except histone mRNA have 3’ poly (A) tail Pre-mRNA is cleaved ~20 nucleotides downstream of polyadenylation signal (upstream- AAUAAA downstream- GU rich (or) simply U rich) ~200- 250 A are added to 3 ´ end

6. Splicing via two transesterification reactions Introns are removed & exons are spliced together Short transcripts- RNA splicing follows cleavage & polyadenylation of 3’ Long transcripts- splicing begins before transcription completes C onsensus sequences 5’ GU- AG 3’ rule , sometimes AU-AC, branch point sequence & polypyrimidine tract

7. Consensus sequences around 5’ and 3’ splice sites in vertebrate pre-mRNAs The central region of the intron, which may range from 40 bases to 500 kilobases in length, generally is unnecessary for splicing to occur.

8. RNA-DNA hybridization studies show that introns are spliced out during pre- mRNA processing

9. Two transesterification reactions that result in splicing of exons in pre-mRNA  In the first transesterification reaction, the 2’ hydroxyl of the branch point A residue attacks the phosphate at the 5’ end of the intron. This releases the 5’ exon and creates a lariat structure within the intron   In the second transesterification reaction, the 3’ hydroxyl of the detached 5’ exon attacks the phosphate at the 3’ end of the intron, resulting in release of the intron lariat and ligation of the exons

10. Model of spliceosome -mediated splicing of pre-mRNA  Splicing RNA splicing occurs in nuclear particles known as spliceosomes S pecificity of splicing comes from the five small snRNP — RNAs denoted U1, U2, U4, U5, and U6 , which contain sequences complementary to the splice junctions

11. Chain elongation by RNA polymerase II is coupled to the presence of RNA- procesing factors Capping enzyme & RNA splicing polyadenylation factors associates with phosphorylated CTD This mechanism may ensure that pre-mRNA is not synthesized unless the machinery for processing it is properly positioned Excised introns are degraded primarily by exosomes - multiprotein complexes that contain 11 3’ to 5’ exonucleases as well as RNA helicases. Exosomes also degrade improperly processed pre-mRNA

12. SR proteins contribute to exon definition in long pre-mRNA Average length of exon app. 150 bases, intron app. 3500 bases SR proteins RNA binding proteins , interacts with exonic splicing enhancers Mediates co-operative binding of U1 snRNP to a true splice site and U2 snRNP to a branch point Cross-exon recognition complex (complex of SR proteins, snRNPs & splicing factor-U2AF)

13. Alternative splicing most pre-mRNAs contain multiple introns, different mRNAs can be produced from the same gene by different combinations of 5' and 3' splice sites J oining of exons in varied combinations controls gene expression by generating multiple mRNAs (and therefore multiple proteins) from the same pre-mRNA About 50% of human genes transcripts ( diversity of proteins encoded by 20,000-25,000 gene ) C an vary in different tissues and in response to extracellular signals Eg ., sex determination in Drosophila

14. Alternative splicing is the primarily mechanism for regulating mRNA processing

15. Processing of rRNA & tRNA 80% rRNA, 15% tRNA, 2-5% mRNA B asic processing is similar in prokaryotic & eukaryotic cells rRNA processing- Nucleolus- not surrounded by a membrane, is associated with chromosomal regions that contain the genes for the 5.8S, 18S, and 28S rRNAs Ribosomes of higher eukaryotes- 4 types of RNA designated the 5S, 5.8S, 18S, and 28S rRNAs

16. Ribosomal RNA Genes and the Organization of the Nucleolus Transcribed as a single unit within the nucleolus by RNA polymerase I , yielding a 45S ribosomal precursor RNA Transcription of the 5S rRNA, takes place outside the nucleolus in higher eukaryotes and is catalyzed by RNA polymerase III 200 copies of the gene- 5.8S , 18S , & 28S rRNAs, approximately 2000 copies of the gene- 5S rRNA

17. genes for 5.8S, 18S, and 28S rRNAs are clustered in tandem arrays on five different human chromosomes ( chromosomes 13, 14, 15, 21, and 22 ) 5S rRNA genes are present in a single tandem array on chromosome 1 Following each cell division, nucleoli become associated with the chromosomal regions that contain the 5.85, 185, and 285 rRNA genes, which are therefore called nucleolar organizing regions Size of the nucleolus depends on the metabolic activity of the cell (granular component)

18. Processing of rRNA takes place within the nucleolus of eukaryotic cells

19. In addition to cleavage, the processing of pre-rRNA involves base modification, addition of methyl groups to specific bases(10) & ribose residues(100) conversion of uridine to pseudouridine (100) processing of pre-rRNA requires the action of both proteins (300) and RNAs (small nucleolar RNAs - snoRNAs )

20. Individual snoRNPs consist of single snoRNAs associated with eight to ten proteins snoRNPs then assemble on the pre-rRNA to form processing complexes Some snoRNAs are responsible for the cleavages of pre-rRNA into 185, 5.85 , and 285 products most abundant nucleolar snoRNA is U3 (200,000 copies per cell) cleaves pre-rRNA within the 5' external transcribed spacer sequences U8 snoRNA cleaves pre-rRNA to 5.85 and 285rRNAs U22 snoRNA cleaves pre-rRNA to 185 rRNA

21. Most snoRNAs , however, function in rRNA synthesis as guide RNAs to direct the specific base modifications of pre-rRNA, including the methylation of specific ribose residues and the formation of pseudouridines Most of the snoRNAs contain short sequences of approximately 15 nucleotides that are complementary to 185 or 285 rRNA-for modification

22. tRNA processing tRNAs in both bacteria and eukaryotes are synthesized as longer precursor molecules (pre-tRNAs) processing of the 5' end of pre-tRNAs involves cleavage by an enzyme called RNase P ( Sidney Altman) 3' end of tRNAs is generated by the action of a conventional protein Rnase addition of a CCA terminus at their 3' ends ( site of amino acid attachment)- CCA terminus is encoded in the DNA of some tRNA genes, but in others it is not.

23. extensive modification of bases in tRNA molecules (app. 10 % ) Some pre-tRNAs, as well as pre-rRNAs in a few organisms, contain introns that are removed by splicing i.e., by endonucleases

24. Differences of pre-tRNA splicing Splicing of pre-tRNA catalyzed by proteins not by RNAs Intron is excised in one step-simultaneous cleavage of both the intron ends GTP/ATP is required

25. Trans-splicing Trypanosomes, euglenoids mRNAs are constructed by splicing together separate RNA molecules Carried out by snRNPs Also seen in 10-15% of mRNA of C. elegans Y shaped intron is released

26. Self splicing RNA can catalyze the removal of their own introns in the absence of other protein or RNA factors Intron acts as a ribozyme described by Tom Cech et.al., during studies of the 285 rRNA of the protozoan Tetrahymena self-splicing RNAs in mitochondria, chloroplasts , and bacteria

27. 2 self splicing RNAs (based on their reaction mechanisms ) G roup I introns (e.g., Tetrahymena pre-rRNA) cleavage at the 5' splice site mediated by a guanosine cofactor. The 3' end of the free exon then reacts with the 3' splice site to excise the intron as a linear RNA. Group II introns (e.g., some mitochondrial pre-mRNAs) closely resemble to nuclear premRNA splicing Indicates active catalytic components of the spliceosome were RNAs rather than proteins- snRNAs (U2, U6)

28. RNA editing Refers to RNA processing events (other than splicing) that alter the protein-coding sequences of some mRNAs. first discovered in mitochondrial mRNAs of trypanosomes also described in mitochondrial mRNAs of other organisms, chloroplast mRNAs of higher plants, and nuclear mRNAs of some mammalian genes .

29. 2 TYPES- Base modification: A to I, C to U (Deamination )- vertebrates Insertion/Deletion: U insertion or deletion RNA editing by the deamination of adenosine to inosine is the most common form of nuclear RNA editing in mammals. This form of editing plays an important role in the nervous system

30. Editing of A polipoprotein B mRNA Tissue-specific editing of Apo-B mRNA thus results in the expression of structrally and functionally different proteins in liver and intestine . The full-length Apo- BlOO (app. 500kDa) produced by the liver transports lipids in the circulation; Apo-B48(app. 250kDa) functions in the absorption of dietary lipids by the intestine. RNA editing by deamination

31. Transcriptional activators, repressors & transcription control

32. Expression of eukaryotic genes is controlled primarily at the level of initiation of transcription & also regulated during elongation Cis-Acting Regulatory Sequences : Promoters and Enhancers Binding sites for regulatory factors, c ontrol the expression of individual adjacent genes Enhancers- sequences stimulate transcription from other promoters as well (SV40), activity depended on neither their distance nor their orientation with respect to the transcription initiation site

33. The activity of enhancer is specific for the promoter of its appropriate target gene. S pecificity is maintained in part by insulators or barrier elements , which divide chromosomes into independent domains and prevent enhancers from acting on promoters located in an adjacent domain

34. Structure and Function of Transcriptional Activators Consist of 2 independent domains DNA-binding domain- recognizes a specific DNA sequence, Activation domain- interacts with Mediator or other components of the transcriptional machinery Eukaryotic cells- about 2500 transcription factors They contain many distinct types of DNA-binding domains

35. DNA-binding domains

36. Zinc finger domain Most common Contains repeats of 2 cysteine & 2 histidine residues that bind zinc ions- common, Identified in the polymerase III transcription factor TFIIIA, polymerase II promoters- Spl S teroid hormone receptors (estrogen & testosterone)- 4 cysteine with central zinc α helix interacts with major groove of DNA

37. Helix-turn-helix motif F irst recognized in prokaryotes- E. coli catabolite activator protein ( CAP) One helix makes most of the contacts with DNA, while the other helices lie across the complex to stabilize the interaction In eukaryotic cells- homeodomain proteins, role in the regulation of gene expression during embryonic development

38. Leucine zipper & Helix-loop-helix proteins C ontain DNA-binding domains formed by dimerization of two polypeptide chains leucine zipper contains 4 or 5 Leucine residues spaced at intervals of seven amino acids Dimerization domain held together by hydrophobic interactions between leucine side chains- Leucine zipper Region rich in positively charged amino acids (lysine and arginine) that binds DNA Helix-loop-helix proteins- dimerization domains formed by 2 helical regions separated by a loop

39. Combination of distinct protein subunits can form an expanded array of factors that can differ both in DNA sequence recognition and in transcription-stimulating activities Activation domains- not well characterized Acidic activation domains- rich in negatively charged residues (aspartate and glutamate); others are rich in proline or glutamine residues

40. Action of transcriptional activators 2 mechanisms: 1 ) they interact with Mediator proteins and general transcription factors to facilitate the assembly of a transcription complex and stimulate transcription 2 ) they interact with coactivators that facilitate transcription by modifying chromatin structure

41. Eukaryotic Repressors Binds to specific DNA sequences and inhibit transcription interfere with the binding of other transcription factors to DNA compete with activators for binding to specific regulatory sequences interacts with corepressors -modifies chromatin structure Binds promoter or enhancer blocks the binding of the activator

42. Action of eukaryotic repressors Active repressors- contain specific functional repressor domains that inhibit transcription via protein-protein interactions Eg ., Kruppel gene repression domain of Kriippel is rich in alanine residues, whereas other are rich in proline or acidic residues.

43. Relationship of Chromatin Structure to Transcription DNA of all eukaryotic cells is tightly bound to histones Basic structural unit of chromatin is the nucleosome Actively transcribed genes- decondensed chromatin Tight winding of DNA around the nucleosome core is major obstacle to transcription, affecting both the ability of transcription factors to bind DNA and the ability of RNA polymerase to transcribe through a chromatin template.

44. Histone acetylation Transcriptionally active chromatin Core histones (H2A, H2B, H3 and H4) have two domains: histone fold domain amino-terminal tail domain (rich in lysine- modified by acetylation) H istone deacetylases , which remove the acetyl groups from histone tails- inhibits transcription

45. Transcriptionally active chromatin- histone H3, including methylation of lysine-4, phosphorylation of serine-10, acetylation of lysines 9, 14, 18, and 23, and methylation of arginines 17 and 26 Deacetylation & methylation of lysines 9, 27, and 36 is associated with repression and chromatin condensation Nucleosome remodeling factor- sliding of histone octamers along the DNA molecule- accessibility of specific DNA sequences to transcription factors

46. DNA Methylation DNA is methylated specifically at the cytosines (C) that precede guanines (G) in the DNA chain ( CpG dinucleotides ), and this methylation is correlated with transcriptional repression. Methylation commonly seen in transposable elements regulatory role of DNA methylation- as genomic imprinting, which controls the expression of some genes involved in the development of mammalian embryos

47. Regulation of Transcription-MicroRNAs ( miRNAs )

48. X Chromosome inactivation Noncoding RNA transcribed from a regulatory gene, called Xist , localized to the inactive X, binding & coats- recruitment of a protein complex that induces methylation of histone H3 lysine-27 and lysine-9, leading to chromatin condensation and conversion of most of the inactive X to heterochromatin.

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