Rna processing

this powerpoint is about the processing of diffent types of Rna's (genetics)

1. WELCOME

2. M RNA PROCESSING PROKARYOTES AND EUKARYOTES K.VIJAYREDDY

3. RNA (Ribonucleic Acid) RNA is much more abundant than DNA There are several important differences between RNA and DNA. The pentose sugar in RNA is ribose, in DNA it’s deoxyribose. In RNA, uracil replaces the base thymine (U pairs with A). RNA is single stranded while DNA is double stranded.

4. Primary structure of RNA A, C, G, and U are linked by 3’-5’ ester bonds between ribose and phosphate.

5. Secondary structure of RNA D A) Single stranded regions formed by unpaired nucleotides B) Duplex double helical RNA (A-form with 11bp per turn) C) Hairpin duplex bridged by a loop of unpaired nucleotides D) Internal loop nucleotides not forming Watson-Crick base pairs E) Bulge loop unpaired nucleotides in one strand, other strand has contiguous base pairing F) Junction three or more duplexes separated by single stranded regions G) Pseudoknot tertiary interaction between bases of hairpin loop and outside bases G A B C D E F

6. Tertiary structure 6 Primary: C ovalent bonds & Secondary/Tertiary Non-covalent bonds(H-bonds)

7. RNA structure: 3 levels of organization 7 D Dobbs ISU - BCB 444/544X: RNA Structure & Function Rob Knight Univ Colorado

8. RNA types & functions 8 Types of RNAs Function mRNA - messenger Template for protein synthesis. rRNA - ribosomal Component of ribosome's (protein synthesis) . t-RNA - transfer Transfer of amino acid (protein synthesis). hnRNA - heterogeneous nuclear Precursors & intermediates of mature mRNAs & other RNAs (Premature mRNA). scRNA - small cytoplasmic Signal Recognition Particle (SRP) tRNA processing . snRNA - small nuclear snoRNA - small nucleolar Participate in the splicing and transfer of hnRNA. rRNA processing/maturation/ methylation . miRNA -micro RNA Usually endogenous, induce degradation of targeted mRNA. that block expression of complementary mRNAs. regulation of transcription and translation. siRNA - small interfering RNA Usually exogenous, induce degradation of targeted mRNA. regulation of transcription and translation. ncRNA -non-coding RNA ( npcRNA , nmRNA , fRNA ) all RNA other than mRNA, functional   RNA  molecule that is not  translated  into a  protein . longer than 200nt.

9. Ribosome's are the sites of protein synthesis. They consist of ribosomal RNA (65%) and proteins (35%). They have two subunits, a large one and a small one. Ribosomal RNA

10. Smallest RNA The existence of tRNA was demonstrated by Hoagland in 1957. Anticodon loop has 3 nucleotides that function as anticodon . . Thymine loop function as the ribosome attachment region. The DHU loop serves as the aminoacyl synthetase recognition region. Base-pairing involving H bonds. Transfer RNA( tRNA )

11. tRNA 3-D structure tRNA has a tertiary structure that is L-shaped

12. Messenger RNA (mRNA) It is a single stranded base for base complementary copy of one DNA strand& provides information for amino acid sequence of a polypeptide. Carries genetic information from nucleus to cytoplasm (Template of protein synthesis). Present in two forms : Active form: actively supports translation. Inactive form: does not support translation. .

13. Three main parts : 5 ’ untranslated region (5’ UTR) or leader sequence Coding sequence, specifies amino acids to be translated 3 ’ untranslated region ( 3’ UTR) or trailer sequence

14. These are catalytic RNAs that mainly participate in the cleavage of RNA. They are not true catalysts because they alter their own structure as a result of catalysis. Example: 1. RNase P is a common ribozyme that matures tRNA that acts as an endonuclease. 2. Self-splicing introns. Clinical applications:- Used as therapeutic agents in correcting mutant mRNA in human cells and inhibiting unwanted gene expression. Kill cancer cells. Prevent virus replication. Gene inhibitors. Gene amenders. Protein inhibitors. Immuno stimulatory RNA’s. Ribozymes

15. Most newly transcribed RNA molecules (primary transcripts) undergo various alterations to yield the mature product. (or) RNA processing is the collective term used to describe the molecular events allowing the primary transcripts to become the mature RNA. (or) The process of modification, mainly through cleavage & or splicing, of primary RNA transcripts so as to release functional RNA molecules from them. It is carried out by ribonucleases ( RNases ) that cleave RNA. What is RNA processing……!

16. These RNases not only process the RNA transcripts, also degrade the tRNA, mRNA, rRNA, and other RNA molecules as a part of the normal cellular “Turnover process”. Turnover process refers to degradation of old molecules and the synthesis of new molecules to replace them. Both exo -and endo ribonuclease participate in the turnover process.

17. P rimary transcript M ature RNA . Nucleus or Nucleolus Cytoplasm RNA processing Removal of nucleotides addition of nucleotides to the 5’- or 3’- ends modification of certain nucleotides

18. 1) Remvoal of nucleotides by both endonucleases and exonucleases . Endonucleases to cut at specific sites within a precursor RNA. Exonucleases to trim the ends of a precursor RNA. 2) Addition of nucleotides to 5’-or 3’-ends of the primary transcripts or their cleavage products. Add a cap and a poly(A) tail to pre- mRNA . AAAAAA 3) Modification of certain nucleotides on either the base or the sugar moiety. Add a methyl group to 2’-OH of ribose in mRNA and rRNA .

19. Processing of mRNA hnRNP snRNP particles 5’Capping 3’Cleavage Polyadenylation Splicing Pre-mRNA methylation mRNA PROCESSING Genetic information is transferred from genes to the proteins they encode via a “messenger” RNA intermediate.

20. Characteristics of the Five RNA Polymerases of Eukaryotes Enzyme Location Products RNA polymerase I Nucleolus Ribosomal RNAs, excluding 5S rRNA RNA polymerase II Nucleus Nuclear pre-mRNAs RNA polymerase III Nucleus tRNAs, 5S rRNA, and other small nuclear RNAs RNA polymerase IV Nucleus (plant) Small interfering RNAs (siRNAs) RNA polymerase V Nucleus (plant) Some siRNAs plus noncoding (antisense) transcripts of siRNA target genes

21. mRNA Processing in prokaryotes There is little or no processing of mRNA transcripts in prokaryotes. In fact, ribosomes can assemble proteins before mRNA molecules have not yet been completely synthesized. Prokaryotic mRNA is degraded rapidly from the 5’-end therefore only be translated for a limited amount of time.

22. DNA Cytoplasm Nucleus Eukaryotic mRNA Transcripts are Processed Export G AAAAAA RNA Transcription Nuclear pores G AAAAAA RNA Processing mRNA The mRNA then moves out of the nucleus and is translated in the cytoplasm.

23. mRNA is synthesized by RNA Pol II as longer precursors (pre-mRNA), the population of different RNA Pol II transcripts are called heterogeneous nuclear RNA (hnRNA). Among hnRNA, those processed to give mature mRNAs are called pre-mRNAs. Pre-mRNA molecules are again processed to give mature mRNAs by 5’-capping, 3’-cleavage and poly adenylation , splicing and methylation. Processing of mRNA

24. hnRNP :- hnRNA + proteins The hnRNA synthesized by RNA Pol II is mainly pre-mRNA and rapidly becomes covered by proteins to form heterogeneous nuclear R ibonucleoprotein (hnRNP ). The hnRNP proteins are keep the hnRNA in a single-stranded form and to assist in the various RNA processing reactions .

25. SnRNP particles : snRNA + proteins Eukaryotic nuclei contain many discreet small RNA species called small nuclear RNAs( snRNAs ) are rich in the base uracil , which complex with specific proteins to form snRNPs . The most abundant snRNP are involved in pre-mRNA splicing , U1,U2,U4,U5 and U6 . snRNAs are synthesized in the nucleus by RNA Pol II and have a normal 5’-cap. .

26. Removes the - phosphate Capping – Step 1

27. Capping –Step 2 Attaches guanosine nucleotide OH OH

28. OH OH Adds methyl groups to guanine & riboses Capping – Step 3 OH O CH 3 7-Methylguanine

29. 5 ¢ 3 ¢ 5 ¢ 5 ¢ 3 ¢ Endonuclease cleavage occurs about 20 nucleotides downstream from the AAUAAA sequence. PolyA-polymerase adds adenine nucleotides to the 3 ¢ end. Polyadenylation sequence PolyA tail AAAAAAAAAAAA .... AAUAAA AAUAAA AAUAAA Cleavage/ Polyadenylation G/U

30. Splicing The process of cutting the pre-mRNA to remove the introns and joining together of the exons is called splicing . It takes place in the nucleus before the mature mRNA can be exported to the cytoplasm. Most genes have their protein-coding information interrupted by non-coding sequences called “introns” . The coding sequences are then called “exons” . Introns: non-coding sequences. Exons: coding sequences.

31. Splicing based on secondary structure:

32. Spliceosome mediated splicing: All the known introns begin with the dinucleotide GT and end with the dinucleotide AG this is known as GT-AG rule The GT dinucleotide depicts the donor splicing site and AG dinucleotide depicts the acceptor splicing site

33. Splicing – Intron Sequences 3 ¢ 5 ¢ 5 ¢ splice site 3 ¢ splice site Branch site Intron Exon Exon Py 12 N PyAGG A / C GGU Pu AGUA UACUUAUCC Exon n ……A G G U A A G U …Intron …Y N Y Y R A Y …....Y 12 N C A G N ….. Exon n+1 64 73 100 100 62 68 84 63 80 80 87 75 100 95 65 100 100 Branch Point Yeast consensus

34. Intron loops out and exons brought closer together Splicing of Pre-mRNAs

35. Splicing of Pre-mRNA This is a Spliceosome Composed of five snRNPs (U1, U2, U4, U5 and U6), other splicing factors, and the pre-mRNA being assembled.

36. Intron will be degraded and the snRNPs used again Splicing of Pre-mRNA

37. All noncoding introns are spliced out of a pre- mRNA by the Spliceosome. But not all exons are included in the final mRNA. mRNA can undergo alternative splicing. The selective inclusion or exclusion of exons occur. From one pre-mRNA can make many different mRNA(thus different proteins) >50% of human genes undergo alternative splicing. ALTERNATIVE SPLICING

38. Alternative Splicing of Tropomyosin * Alternative pA * * Alternative pA *

39. However, multiple introns may be spliced differently in different circumstances, for example in different tissues. 1 2 3 5 Heart muscle mRNA 1 4 3 5 Uterine muscle mRNA Thus one gene can encode more than one protein. The proteins are similar but not identical and may have distinct properties. This is important in complex organisms 3 5 4 2 1 pre-mRNA

40. Sex in Drosophila is largely determined by alternative splicing

41. RNA editing This is a form of RNA processing in which the nucleotide sequence of the primary transcript is altered by either Changing residues, D eleting residues, Inserting residues at specific points along the molecule Changing RNA sequence (after transcription).

42. Apolipoprotein -B mRNA in mammalian intestine and liver The mammalian liver cells contain apolipoprotein - B having 4563 amino acids. While in intestine cells this protein has only 2152 amino acids. In intestine cells, the codon 2153 is modified in the mRNA , the C of this codon , CAA, is changed to U to give rise to the codon UAA, which causes chain termination. A guide RNA containing a sequence that is complementary to the correctly edited mRNA provides a mechanism of U insertion or deletion.

43. Liver Intestine

44. mRNA processing: overview

45. Possible questions: Describe in detail about mRNA processing ? Difference between splicing and alternate splicing? RNA editing?

46. REFERNCES: GENETICS- B.D.SINGH PRINCIPLES OF GENETICS- SNUSTARD AND SIMMONS

47. Thank U