1. RNA PROCESSING AND RNPs
Molecular Biology Course
Section O
RNA PROCESSING AND RNPs

2. 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.

3. 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.

4. endonucleases to cut at specific sites within a precursor RNA
(1) Removal 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
This general process is seen in prokaryotes and eukaryotes for all types of RNA

5. (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

6. Add a methyl group to 2’-OH of ribose in mRNA (A) and rRNA
(3) Modification of certain nucleotides on either the base or the sugar moiety.
Add a methyl group to 2’-OH of ribose in mRNA (A) and rRNA
Extensive changes of bases in tRNA

7. RNPs Ribonucleoproteins = RNA protein complexes
The RNA molecules in cells usually exist complexed with proteins
specific proteins attach to specific RNAs
Ribosomes are the largest and most complex RNPs

8. 3-D structure

9. Digital cryo-electron micrography
RNP颗粒的低温电镜图

10. O1: rRNA PROCESSING AND RIBOSOMES
Section O: RNA processing and RNPs
O1: rRNA PROCESSING AND RIBOSOMES
rRNA processing in prokaryotes
rRNA processing in eukaryotes
Prokaryotic ribosomes
Eukaryotic ribosomes

11. O1-1: rRNA processing in prokaryotes
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.

12. O1-1: rRNA processing in prokaryotes
3. The initial transcript has a sedimentation coefficient of 30s (6000 nt) and is normally quite short-lived(fig1)
Pre-16S rRNA
Pre-tRNA
Pre-23S rRNA
pre-5S rRNA
Promoters
Terminators
rRNA operon

13. O1-1: rRNA processing in prokaryotes
RNase III, involved in the first step of rRNA processing
RNase M5, M16 and M23 are involved in the second step of rRNA processing

14. O1-1: rRNA processing in prokaryotes
Pre-16S rRNA
Pre-tRNA
Pre-23S rRNA
pre-5S rRNA
Promoters
Terminators
Transcription
30S pre-rRNA
Processing steps

15. 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

16. 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

17. 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

18. O1-1: rRNA processing in prokaryotes: CLEAVAGE
rRNA operon
Pre-16S rRNA
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

19. O1-2: 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

20. O1-2: rRNA processing in Eukaryotes: rRNA features
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.

21. 3. The precursor contains one copy of the 18S coding region and
O1-2: rRNA processing in Eukaryotes: rRNA features
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.

22. the transcript undergoes little or no processing
O1-2: rRNA processing in Eukaryotes: rRNA features
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

23. 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

24. Mature rRNAs complex with protein to form RNPs (nucleolus)
O1-2: rRNA processing in Eukaryotes: rRNA processing
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)

25. O1-2: rRNA processing in Eukaryotes: rRNA processing
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

26. O1-3: 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.

27. Prokaryotic Ribosome
E. coli ribosome is 25 nm in diameter, 2750 kD in mass, and consists of two unequal subunits that dissociate at < 1mM Mg2+

28. Prokaryotic Ribosomal proteins Size varied from 46 aa to 557 aa.
Prokaryotic Ribosome
Prokaryotic Ribosomal proteins
Size varied from 46 aa to 557 aa.
Most are basic proteins which bind to RNA.
rRNA chaperons to assist the folding of rRNA to catalytic structure.
Ribosomal rRNA
Responsible for peptide bond formation.

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

30. Prokaryotic Ribosome
Ribosome Assembly
Assembly is coupled w/ transcription and pre-rRNA processing

32. Prokaryotic Ribosome
Ribosome Structure (1)

33. 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.

34. Eukaryotic Ribosome
larger and more complex than prokaryotic ribosomes, but with similar structural and functional properties

35. O2: tRNA PROCESSING, RNase P AND RIBOZYMES
Section O: RNA processing and RNPs
O2: tRNA PROCESSING, RNase P AND RIBOZYMES
tRNA processing in prokaryotes
tRNA processing in eukaryotes
RNase P
Ribozymes

36. tRNA 3-D structure

37. 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.

38. tRNA processing in prokaryotes
Primary transcripts
RNase D,E,F and P
(See your text book)
tRNA with mature ends
Base modifications
mature tRNAs

39. tRNA processing in eukaryotes
The pre-tRNA is synthesized with a
16 nt 5’-leader,
a 14 nt intron and
two extra 3’-nucleotides.

41. tRNA processing in Eukaryotes
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

42. RNase P
Ribonuclease P (RNase P) is an enzyme involved in tRNA processing that removes the 5' leader sequences from tRNA precursors

43. RNase P (2)
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.

44. RNase P (1)
RNA component can catalyze pre-tRNA in vitro in the absence of protein. Thus RNase P RNA is a catalytic RNA, or ribozyme.

45. Ribozyme (1)
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.

46. Ribozyme (2)
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.

47. Ribozyme (3)
Self-cleaving RNA encoded by viral genome to resolve the concatameric molecules of the viral genomic RNA
HDV ribozyme
Hairpin ribozyme
Hammer head ribozyme

48. Ribozyme (4) Ribozymes can be used as therapeutic agents in
correcting mutant mRNA in human cells
inhibiting unwanted gene expression
Kill cancer cells
Prevent virus replication

49. O3: mRNA PROCESSING, hnRNPs AND snRNPs
Section O: RNA processing and RNPs
O3: mRNA PROCESSING, hnRNPs AND snRNPs
Processing of mRNA
hnRNP
snRNP particles
5’Capping
3’Cleavage and polyadenylation
Splicing
Pre-mRNA methylation

50. 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

51. 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

52. Processing of mRNA in eukaryotes
Pre-mRNA molecules are processed to mature mRNAs by 5’-capping, 3’-cleavage and polyadenylation, splicing and methylation.

53. Eukaryotic mRNA processing: overview

54. hnRNP: hnRNA + proteins
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

55. snRNP particles: snRNA + proteins
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.

56. snRNP particles
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.

57. 5’ Capping Processing of mRNA in eukaryotes
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

58. 7-methylguanosine (m7G)

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

60. 3’ Cleavage and polyadenylation
Processing of mRNA in eukaryotes
3’ 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.

61. 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

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

64. Processing of mRNA in eukaryotes
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.

65. Introns: non-coding sequences Exons: coding sequences
Processing of mRNA in eukaryotes
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)

66. Biochemical steps of pre-mRNA splicing
Processing of mRNA in eukaryotes
Biochemical steps of pre-mRNA splicing
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.

67. Lariat
C U R A Y

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

69. Spliceosome Catalyzes pre-mRNA splicing in nucleus
Processing of mRNA in eukaryotes
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.

72. Splicing cycle

73. Processing of mRNA in eukaryotes
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.

75. O4: ALTERNATIVE mRNA PROCESSING
Alternative processing
Alternative poly(A) sites
Alternative splicing
RNA editing

76. 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.

77. Alternative poly(A) sites
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.

78. 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

79. Alternative splicing

80. Alternative splicing

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

82. (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.

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

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

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

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

87. Sex in Drosophila is largely determined by alternative splicing

88. Sxl-Sex lethal gene: the master regulatory gene at the top of the sex determination pathway. The downstream targets of the Sxl protein include transcripts from the Transformer (Tra) and Male-specific lethal 2 (Msl2) genes. Tra gene also encodes a splicing regulator.

89. 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)

90. 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)

91. Deamination in mammalian
Apolipoprotein-B mRNA in mammalian intestine and liver. The genome contains a single (interrupted) gene whose sequence is identical in all tissues, with a coding region of 4563 codons.
a protein of 512 kD representing the full coding sequence is found in the liver.
A shorter form of the protein, ~250 kD, is synthesized in intestine. This protein consists of the N-terminal half of the full-length protein, which is caused by the change of a CAA codon to a UAA. (see Figure)

92. Liver
Intestine

93. Deamination in mammalian 2. glutamate receptors in rat brain.
Editing at one position changes a glutamine codon in DNA into a codon for arginine in RNA (AI)
The change affects the conductivity of the channel and therefore has an important effect on controlling ion flow through the neurotransmitter.
Deamination is catalyzed by cytidine or adenosine deaminases.
Editing enzymes are related to the general deaminases, but have other regions or additional subunits that control their specificity.

94. Insertion or deletion of U in protozoa
Example: part of the mRNA sequence of Trypanosome brucei coxIII shows many uridines that are not coded in the DNA (shown in red) or that are removed from the RNA (shown as T).

95. Insertion or deletion of U in protozoa
A guide RNA containing a sequence that is complementary to the correctly edited mRNA provides a mechanism of U insertion or deletion. (See figure)