Transcript 5` Capping

RNA Processing
Processing and Export of RNA
Overview
In eukaryotes, the growing functional complexity and cellular compartmentalization
requires that RNA be processed for long term storage and transport through nuclear
membrane.
1. rRNA porcessing
2. tRNA porcessing
3. mRNA porcessing
• 5’m caps
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3’polyA tail
Splicing
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Spliceosomes
Self splicing RNAs
Mutations in splicing
Alternative splicing
4. RNA editing
5. RNA export
Processing of rRNA Transcripts
• rRNA constitutes 95% of the total RNA in the cell.
• Multiple copies of rRNA genes are tendamly arranged, separated by spacer regions.
• The pattern of alternating transcribed and non-transcribed region is readily obvious.
Ribosomal RNA Processing
• The rRNAs are named according to their "S" values,
which refer to their rate of sedimentation in an
ultra-centrifuge.
• The larger the S value, the larger the rRNA.
• 45S precursor rRNA is chemically modified and
enzymatically cleaved in to smaller S fragments.
• Two types of chemical modifications are added:
• Psueodouridine
• 2’O methyl ribose.
• The spacer sequences between 18S, 5.8S and 28S
rRNA are discarded and degraded.
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tRNA Processing
• A tRNA molecule consists of 70-80 nucleotides.
• Some nucleotides in tRNA are modified to:
• dihydrouridine (D),
• pseudouridine (Y),
• inosine (I),
• Inosine plays an important role in codon recognition.
• In addition to these modifications, a few nucleosides are methylated.
• The major role of tRNA is to translate mRNA sequence into amino acid sequence.
Structure of Mature Eukaryotic mRNA
• All mRNAs have a 5’ cap
• All mRNA (except histone) have a 3’ poly(A) tail.
• the 5’ cap and 3’ poly(A) tail prevent mRNA degradation.
Messenger RNA Processing
Overview:
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5’-Capping
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3’-polyadenylation
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Splicing
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Splice site recognition
Spliceosomes
Self splicing RNAs
Mutations that disrupt splicing
Alternative splicing
5’ Capping
• Capping occurs co-transcriptionally.
• 7-methly guanosine (7mG) cap is added to the 5’ end.
• Guanyltransferase forms a 5'-5' linkage between
7-methyl guanosine and 5’-PO4 of the first base.
• This methyl group is called Cap0.
• O-methyl transferase then adds a methyl group to
the 2’-OH of the first two original bases in RNA.
• These methyl groups are referred to as CAP1 and CAP2.
• Methyl groups protect mRNA from degradation.
5’-Methylated Caps in mRNA
• Methylated bases in caps 0 and 1.
• m7G-5'-p-p-p-5'-Adenosine-3'-p-etc
• O-Methylated sugars in caps 1 and 2.
3’-Polyadenylation of mRNA
Pimary RNA transcript is cleaved and modified by RNA specific proteins.
Cleavage and polyA signal is located ~ 30 residues upstream of a GU rich
termination site.
3’-Poly-Adenylation of mRNA
• An Endonuclease cleaves the primary transcript at CA,
~10-30 nucleotides downstream of the AAUAAA.
• Poly-A polymerase then adds ~ 200 adenosine residues.
• Poly-A tail is associated with Poly-A binding proteins.
• Poly-A tails stabilize mRNA.
RNA can fold into specific structures
(A) Diagram of a folded RNA structure showing only conventional base-pair
interactions; (B) structure with both conventional (red) and nonconventional
(green) base-pair interactions; (C) structure of an actual RNA. Each conventional
base-pair interaction is indicated by a "rung" in the double helix. Bases in other
configurations are indicated by broken rungs.
Splicing of mRNAs
• Coding sequences in eukaryotic RNA is frequently interrupted by non-coding regions.
• Removal of intervening sequences or Introns (grey) from pre-mRNA transcripts.
• Joining of the expressed sequences or Exons (color) to form mature mRNA transcripts.
• Splicing occurs in the nucleus before transport to the cytoplasm.
• cDNA contain only exons.
• Genomic DNAs contain both exons and introns.
• Incidence of introns increases with complexity in organisms.
Variation in intron and exon lengths
in the human, worm, and fly genomes
A) Size distribution of exons. (B) Size distribution of introns. Note that
exon length is much more uniform than intron length. (Adapted from
International Human Genome Sequencing Consortium, Nature
409:860 921, 2001.)
Heteronuclear Ribonucleoproteins (hnRNPs)
Self Hybridization of RNA molecules in vitro is prevented by hnRNP proteins.
The presence of complex secondary structures within RNA molecules inhibits
hybridization between long complementary sequences in separate molecules.
Association of hnRNP proteins with RNA is thought to prevent formation of RNA
secondary structures, thereby facilitating base-pairing between different
complementary molecules during splicing events. These proteins may have a similar
function in vivo.
Splicing signals
Splice Junction Consensus Sequence :
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In over 60% of cases, the upstream exon sequence is (A/C)AG at the donor site,
and G at the downstream splice acceptor exon site.
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5’-Donor site: AG | GUAAGU, where GU is always at 5' end of intron
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3’-Acceptor site: (Py...Py) 12 NCAG | N, where AG is always at 3' end of intron.
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2’- Branch site: CU(A/G)A(C/U) where A is conserved in all organisms.
The consensus Splice Signal Sequences in an RNA
molecule
Only the three blocks of nucleotide sequences shown are required to remove an intron sequence;
the rest of the intron can be occupied by any nucleotide. Here A, G, U, and C are the standard
RNA nucleotides; R stands for either A or G; Y stands for either C or U. The A highlighted in red
forms the branch point of the lariat produced by splicing. Only the GU at the start of the intron
and the AG at its end are invariant nucleotides in the splicing consensus sequences. The
remaining positions (even the branch point A) can be occupied by a variety of nucleotides,
although the indicated nucleotides are preferred. The distances along the RNA between the three
splicing consensus sequences are highly variable; however, the distance between the branch point
and 3’ splice junction is typically much shorter than that between the 5’ splice junction and the
Splicing of Eukaryotic pre-mRNA
2-Step Trans-esterification:
• Cleavage at the 5'-end (G) of the intron by attack
of a specific 2'OH group (A), the branch site.
• This forms a 2’-5’phosphodiester bond with the 5'phosphate of the intron, creating a lariat structure.
• Ligation of the 3'-OH on Exon 1 with the 5‘phosphate of Exon 2 then releases the lariat (RNA)
intron.
Splicing Branch Point
The structure of the branch point in the lariat
intermediate in which the adenylate residue is
joined to three nucleotides by phosphodiester
bonds.
The new 2’-to-5’ linkage is shown in red, and
the usual 3’-to-5’ linkages are shown in blue.
Spliceosome mediated Splicing
• In most eukaryotes, splicing is mediated by a large ribonucleoprotein complex,
the spliceosome.
• The spliceosome contains a specific set of U-rich small nuclear RNAs associated with
ribonucleoproteins or snRNPs (snurps).
• Splicing snRNPs:
• U1: 5'- site recognition
• U2: branch site recognition
• U4: forms base paired complex and acts with U6
• U5: 3'- junction binding of U4-U6 complex
• U6: complex with U4 makes up the spliceosome transesterase.
• The common spliceosome recognizes introns starting with 5'-GU and ending in AG-3‘
donor (5’) splice site
branch site
acceptor (3’) splice site
G/GUAAGU..................…A.......…YYYYYNYAG/G
U1
U2
Pre-splicing Complex
Interactions between pre-mRNA, U1 snRNA, and U2 snRNA early in the pre-splicing
process. The 5’region of U1 snRNA initially base-pairs with nucleotides at the 5’end of the
intron (blue) and 3’end of the 5’exon (dark red) of the pre-mRNA; U2 snRNA base-pairs with a
sequence that includes the branch-point A, although this residue is not base-paired.
Secondary structures in the snRNAs that are not altered during splicing are shown in
diagrammatic line form.
The purple rectangles represent sequences that bind snRNP proteins recognized by anti-Sm
antibodies. For unknown reasons, antisera from patients with the autoimmune disease systemic
lupus erythematosus (SLE) contain these antibodies. Such antisera have been useful in
characterizing components of the splicing reaction.
snRNP Spliceosome
• Pre-splicing complex is formed by U1 and U2.
• Tri snRNP U5, U4:U6 is then recruited.
• U4 is base paired with U6.
• U5 then displaces U1 from the upstream exon.
• U6 then base-pairs to U2, resulting in displacemant of U4.
• Finally, U5 base pairs to exon 2 near the 3'- splice site on
the same stem loop that already holds Exon1.
• This brings the 3'-OH of Exon 1 into close proximity to
5'-p of exon 2.
• Nucleophilic attack of the phosphodiester bond
completes the splicing process, releasing the intron as
a lariat carrying the various splicing factors.
Self-splicing Introns
Self Splicing Catalytic RNAs
Lower eukaryotes (ribozymes) and mitochondrial RNAs can undergo
self splicing without the help of other splicing factors.
Group I introns
Group II Introns
Secondary structures of group II self-splicing
introns (a) and U snRNAs present in the
spliceosome
The first transesterification reaction is indicated by black arrows; the second reaction, by blue
arrows. The branch-point A is boldfaced. The similarity in these structures suggests that the
spliceosomal snRNAs evolved from group II introns, with the trans-acting snRNAs being
functionally analogous to the corresponding domains in group II introns