1. Section O – RNA processing and RNPs

2. Contents O1 rRNA processing and ribosomes
Types of RNA processing, rRNA processing in prokaryote, rRNA processing in eukaryotes, RNPs and their study, Prokaryotic ribosomes, Eukaryotic ribosomes
O2 tRNA processing, RNase P and ribozymes
tRNA processing in prokaryotes, tRNA processing in eukaryotes, RNase P, Ribozymes
O3 mRNA processing, hnRNPs and snRNPs
Processing of mRNA, hnRNP, snRNP particles, 5’ Capping, 3’ Cleavage and polyadenylation, Splicing, Pre-mRNA methylation
O4 Alternative mRNA processing
Alternative processing, Alternative poly(A) site, Alternative splicing, RNA editing

3. O1 rRNA processing and ribosomes — Types of RNA processing
Very few RNA molecules are transcribed directly into the final mature RNA. Most newly transcribed RNA molecules (primary transcripts) undergo various alterations to yield the mature product. RNA processing is the collective term used to describe the molecular events allowing the primary transcripts to become the mature RNA.

4. primary transcript mature RNA. Cytoplasm Nucleus or Nucleolus
RNA processing
Romoval of nucleotides
addition of nucleotides to the 5’- or 3’- ends
modification of certain nucleotides
mature RNA.

5. (1) Removal of nucleotides by both endonucleases and exonucleases
(2) Addition of nucleotides to 5’-or 3’-ends of the primary transcripts or their cleavage products.
(3) Modification of certain nucleotides on either the base or the sugar moiety.

6. O1 rRNA processing and ribosomes — rRNA processing in prokaryote
There are 7 different operons for rRNA that are dispersed throughout the genome.
Each operon contains one copy of each of the 5S,the 16S and the 23S rRNA sequences. About 1~4 coding sequences for tRNA molecules are also present in these rRNA operons.
The initial transcript has a sedimentation coefficient of 30s (6000 nt) and is normally quite short-lived.
Pre-16S rRNA
Pre-tRNA
Pre-23S rRNA
pre-5S rRNA
Promoters
Terminators
rRNA operon

7. Step 1: Following or during the primary transcription, the RNA folds up into a number of stem-loop structures by base pairing between complementary sequences
RNA folding

8. Step 2: The formation of this secondary structure of stems and loops allows some proteins to bind to form a RNP complex which remain attached to the RNA and become part of the ribosome
RNP complex
formation

9. Step 3: After the binding of proteins, nucleotide modifications take place.
Example: methylation of adenine by methylating agent S-Adenosylmethonine (SAM)
Step 4: RNA cleavage

10. rRNA operon pre-5S rRNA 30S pre-rRNA: Transcription Cleavage at
Pre-tRNA
Pre-23S rRNA
pre-5S rRNA
Promoters
Terminators
30S pre-rRNA:
Transcription
Cleavage at
16S rRNA
tRNA
23S rRNA
5S rRNA
RNase III III P F III III P F P E
RNase M M M M23 M5

11. O1 rRNA processing and ribosomes — rRNA processing in eukaryotes
rRNA in eukaryotes is also generated from a single, long precursor molecule by specific modification and cleavage steps
The processes are not so well understood

12. The rRNA genes are present in a tandemly repeated cluster containing 100 or more copies of the transcription unit, and are transcribed in nucleolus by RNA Pol I
Precursor sizes are different among organisms (yeast: 7000 nt; mammalian nt), and pre-mRNA processing is also slightly different among organism.

13. 3. The precursor contains
one copy of the 18S coding region and
one copy each of the 5.8S and 28S coding regions, which together are the equivalent of the 23S rRNA in prokaryote
4. The large precursor RNA undergoes a number of cleavages to yield mature RNA and ribosome.

14. 5. The eukaryotic 5S rRNA
is transcribed by RNA Pol III from unlinked genes to give a 121nt transcript
the transcript undergoes little or no processing

15. Mammalian pre-rRNA processing Indicates RNase cleavage
ETS1
ITS1
ITS2
ETS2
47S
45S
41S
20S and 32S
Mature rRNAs
18S rRNA
5.8S rRNA
28S rRNA
Mammalian pre-rRNA processing
Indicates RNase cleavage

16. The 5.8S region must base-pair to the 28S rRNA before the mature molecules are produced.
Mature rRNAs complex with protein to form RNPs (nucleolus)
Methylation occurs at over 100 sites to give 2’-O-methylribose, which is known to be carried out by snRNPs (nucleolus)

17. Introns (group I) in rRNA genes of some lower eukarytes (Tetrahymena thermophila) must be spliced out to generate mature rRNAs.
Many group I introns are found to catalyze the splicing reaction by itself in vitro, therefore called ribozyme

19. O1 rRNA processing and ribosomes — RNPs and their study
Cells contain a variety of RNA-protein complexes( RNPs).
These can be studied using techniques that help to clarify their structure and function.
These include dissociation, re-assembly, electron microscopy, use of antibodies, RNase protection, RNA binding, cross-linking and neutron and X-ray diffraction.
The structure and function of some RNPs are quite well characterized.

20. O1 rRNA processing and ribosomes — Prokaryotic ribosomes
Protein biosynthetic machinery
Made of 2 subunits (bacterial 30S and 50S, & Eukaryotes 40S and 60S)
Intact ribosome referred to as 70S ribosome in Prokaryotes and 80S ribosome in Eukaryotes
In bacteria, 20,000 ribosomes per cell, 25% of cell's mass.

22. Features of the E.coli ribosome
Cleft
Platform
Central protuberance
Stalk
Small

23. Ribosome Structure (1)

24. Ribosome Structure (2) mRNA is associated with the 30S subunit
Two tRNA binding sites (P and A sites) are located in the cavity formed by the association of the 2 subunits.
The growing peptide chain threads through a “tunnel” that passes through the 30S subunit.

25. O1 rRNA processing and ribosomes — Eukaryotic ribosomes
larger and more complex than prokaryotic ribosomes, but with similar structural and functional properties

27. O2 tRNA processing, RNase P and ribozymes — tRNA processing in prokaryotes
Mature tRNAs are generated by processing longer pre-tRNA transcripts, which involves
specific exo- and endonucleolytic cleavage by RNases D, E, F and P (general) followed by
base modifications which are unique to each particular tRNA type.

29. O2 tRNA processing, RNase P and ribozymes — tRNA processing in eukaryotes
The pre-tRNA is synthesized with a 16 nt 5’-leader a 14 nt intron and two extra 3’-nucleotides.

30. Primary transcripts forms secondary structures recognized by endonucleases
5’ leader and 3’ extra nucleotide removal
tRNA nucleptidyl transferase adds 5’-CCA-3’ to the 3’-end to generate the mature 3’-end
Intron removal

31. O2 tRNA processing, RNase P and ribozymes — RNase P
Ribonuclease P (RNase P) is an enzyme involved in tRNA processing that removes the 5' leader sequences from tRNA precursors
RNase P enzymes are found in both prokaryotes and eukaryotes, being located in the nucleus of the latter where they are therefore small nuclear RNPs (snRNPs)
In E. coli, the endonuclease is composed of a 377 nt RNA and a small basic protein of 13.7kDa.
RNA component can catalyze pre-tRNA in vitro in the absence of protein. Thus RNase P RNA is a catalytic RNA, or ribozyme.

32. O2 tRNA processing, RNase P and ribozymes — Ribozymes
Ribozymes are catalytic RNA molecules that can catalyze particular biochemical reactions.
RNase P RNA is a ribozyme.
RNase P RNA from bacteria is more catalytically active in vitro than those from eukaryotic and archaebacterial cells. All RNase P RNAs share common sequences and structures.
Self-splicing introns: the intervening RNA that catalyze the splicing of themselves from their precursor RNA, and the joining of the exon sequences
Group I introns, such as Tetrahymena intron
Group II introns.

33. Self-cleaving RNA encoded by viral genome to resolve the concatameric molecules of the viral genomic RNA
HDV ribozyme
Hairpin ribozyme
Hammer head ribozyme
Ribozymes can be used as therapeutic agents in
correcting mutant mRNA in human cells
inhibiting unwanted gene expression
Kill cancer cells
Prevent virus replication

34. O3 mRNA processing, hnRNPs and snRNPs — Processing of mRNA
Processing of mRNA: prokaryotes
There is essentially no processing of prokaryotic mRNA, it can start to be translated before it has finished being transcribed.
Prokaryotic mRNA is degraded rapidly from the 5’ end
Processing of mRNA in eukaryotes
In eukaryotes, 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

35. Eukaryotic mRNA processing: overview

36. O3 mRNA processing, hnRNPs and snRNPs — hnRNP
The hnRNA synthesized by RNA Pol II is mainly pre-mRNA and rapidly becomes covered by proteins to form heterogeneous nuclear ribonucleoprotein (hnRNP)
The hnRNP proteins are though to help keep the hnRNA in a single-stranded form and to assist in the various RNA processing reactions

37. O3 mRNA processing, hnRNPs and snRNPs — snRNP particles
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.
A large number of snRNP define methylation sites in pre-rRNA.
snRNAs are synthesized in the nucleus by RNA Pol II and have a normal 5’-cap.
Exported to the cytoplasm where they associate with the common core proteins and with other specific proteins.
Their 5’-cap gains two methyl groups and then imported back into the nucleus where they function in splicing.

38. O3 mRNA processing, hnRNPs and snRNPs — 5’ Capping
Very soon after RNA Pol II starts making a transcript, and before the RNA chain is more then nt long, the 5’-end is chemically modified.
7-methylguanosine is covalently to the 5´ end of pre-mRNA.
Linked 5´  5´
Occurs shortly after initiation

39. 7-methylguanosine (m7G)

40. Function of 5´cap Protection from degradation
Increased translational efficiency
Transport to cytoplasm
Splicing of first exon

42. RNA polymerase II does not usually terminate at distinct site
O3 mRNA processing, hnRNPs and snRNPs — ’ Cleavage and polyadenylation
In most pre-mRNAs, the mature 3’-end of the molecule is generated by cleavage followed by the addition of a run, or tail, of A residues which is called the poly(A) tail.
RNA polymerase II does not usually terminate at distinct site
Pre-mRNA is cleaved ~20 nucleotides downstream of polyadenylation signal (AAUAAA)
~250 AMPs are then added to the 3´ end
Almost all mRNAs have poly(A) tail

44. Function of poly(A) tail
Increased mRNA stability
Increased translational efficiency
Splicing of last intron

45. O3 mRNA processing, hnRNPs and snRNPs — 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.
Introns: non-coding sequences
Exons: coding sequences
RNA splicing: removal of introns and joining of exons
Splicing mechanism must be precise to maintain open reading frame
Catalyzed by spliceosome (RNA + protein)

46. Biochemical steps of pre-mRNA splicing

47. Step 1: a cut is made at the 5′splice site, separating the left exon and the right intron-exon molecule. The right intron-exon molecule forms a lariat, in which the 5′terminus of the intron becomes linked by a 5′-2′ bond to a base within the intron. The target base is an A in a sequence that is called the branch site
Step 2: cutting at the 3′ splice site releases the free intron in lariat form, while the right exon is ligated (spliced) to the left exon.

48. Lariat
C U R A Y

49. Nuclear splicing occurs by two transesterification reactions in which a free OH end attacks a phosphodiester bond.

50. Spliceosome Catalyzes pre-mRNA splicing in nucleus
Composed of five snRNPs (U1, U2, U4, U5 and U6), other splicing factors, and the pre-mRNA being assembled
U1 binds to the 5’ splice site, then U2 to the branchpoint, then the tri-snRNP complex of U4, U5 and U6. As a result, the intron is looped out and the 5’- and 3’ exon are brought into close proximity.
U2 and U6 snRNA are able to catalyze the splicing reaction.

53. Splicing cycle

54. O3 mRNA processing, hnRNPs and snRNPs — Pre-mRNA methylation
The final modification or processing event that many pre-mRNAs undergo is specific methylation of certain bases.
The methylations seem to be largely conserved in the mature mRNA.

55. O4 Alternative mRNA processing — Alternative processing
Alternative mRNA processing is the conversion of pre-mRNA species into more than one type of mature mRNA.
Types of alternative RNA processing include alternative (or differential) splicing and alternative (or differential) poly(A) processing.

56. O4 Alternative mRNA processing — Alternative poly(A) site
Some pre-mRNAs contain more than one poly(A) site and these may be used under different circumstances to generate different mature mRNAs.
In one cell the stronger poly(A) site is used by default, but in other cell a factor may prevent stronger site from being used.

57. O4 Alternative mRNA processing — Alternative splicing
The generation of different mature mRNAs from a particular type of gene transcript can occur by varying the use of 5’- and 3’- splice sites in four ways:
By using different promoters
By using different poly(A) sites
By retaining certain introns
By retaining or removing certain exons

58. Alternative splicing

59. (A) A cassette exon can be either included in the mRNA or excluded.

60. (B) Mutually exclusive exons occur when two or more adjacent cassette exons are spliced such that only one exon in the group is included at a time.

61. (C, D) Alternative 5’ and 3’ splice sites allow the lengthening or shortening of a particular exon.

62. (E, F) Alternative promoters and alternative poly(A) sites switch the 59- or 39-most exons of a transcript.

63. (G) A retained intron can be excised from the pre-mRNA or can be retained in the translated mRNA.

64. (H) A single pre-mRNA can exhibit multiple sites of alternative splicing using different patterns of inclusion.

65. Alternative splicing

66. O4 Alternative mRNA processing — RNA editing
An unusual form of RNA processing in which the sequence of the primary transcript is altered is called RNA editing.
Changing RNA sequence (after transcription)

67. RNA editing is known to occur in two different situations, with different causes.
In mammalian cells there are cases in which a substitution occurs in an individual base in mRNA, causing a change in the sequence of the protein that is coded. (Base modification:A or C deamination)
In trypanosome mitochondria, more widespread changes occur in transcripts of several genes, when bases are systematically added or deleted. (Base U insertion and deletion)

69. Multiple choice questions
1. Which of the following terms correctly describe parts of the E. coli large (50S) subunit?
A stalk central protuberance valley and cleft.
B upper third lower third valley and stalk.
C cleft valley stalk and small protuberance.
D stalk polypeptide exit site valley and central protuberance.
2. Which ribonucleases are involved in producing mature tRNA in E. coli?
A RNases A, D, E and F.
B RNases D, E, F and H.
C RNases D, E, F and P.
D RNases A, D, H and P.

70. 3. Most eukaryotic pre-mRNAs are matured by which of the following modifications to their ends?
A capping at the 3’-end cleavage and polyadenylation at the 5'-end.
B addition of a GMP to the 5'-end，cleavage and polyadenylation to create the 3'-end.
C addition of a guanine residue to the 5'-end cleavage and polyadenylation to create the 3'-end.
D addition of a GMP to the 5'-end，polyadenylation，then cleavage to create the 3'-end.
4. Which one of the following statements correctly describes the splicing process undergone by most eukaryotic pre-mRNAs?
A in a two-step reaction, the spliceosome removes the exon as a lariat and joins the two introns together.
B splicing requires conserved sequences which are the 5ιsplice site，the 3' -splice site the branch-point and the polypurine tract.
C the U1 snRNP initially binds to the 5'-splice site，U2 to the branchpoint sequence and then the tri-snRNP, U4, US and U6 can bind.
D in the first step of splicing the G at the 3'-end of the intron is joined to the 2’-hydroxyl group of the A residue of the branchpoint sequence to create a lariat.

71. THANK YOU !