RNA PROCESSING

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RNA PROCESSING BCH 6415 Dr. Yang Spring
2006 Part I
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5 Capping of mRNA
RNA triphosphatase
In mammals, single bifunctional capping enzyme
RNA guanylyl transferase
RNA methyltransferase
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• 5 mRNA capping in eukaryotes
• Protects mRNA from ribonucleases mRNAs without
  caps rapidly degraded by 5 ? 3 exonuclease
• Directs mRNA for processing and transport to
  cytoplasm
• Regulates protein synthesis by promoting
  efficient initiation of translation
• Only pol II transcripts are capped ?probably due
  to association of capping and transcription
  machineries
• Yeast capping enzymes (guanylyltransferase
  methyltransferase) are bound to CTD of pol II
• Capping enzyme binds only to phosphorylated form
  of CTD
• Capping of pre-mRNA occurs early in
  transcription
• In Drosophila, majority of nascent mRNAs 30 nt
  long already capped

Proudfoot et al. Cell 108501 2002
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SPT5 binds to unphos. form of pol II interacts
with capping enz. released after CTD
phosphorylation
Capping enz. stimulated by phosphorylated CTD
and SPT5
Importin-a stimulates methyltransferase
a, Pol II A form containing unphosphorylated CTD
initiates transcription, produces 2025
nucleotide 5' triphosphorylated transcripts, and
pauses with SPT5 (see refs 18, 19) bound as part
of a large transcription complex. b, CTD is
phosphorylated in the O form, changes
conformation and binds capping enzyme (CE). CE
modifies the exposed end of the nascent
transcript, stimulated by SPT5 as well as P-CTD.
MT binds to CE (mammals) or P-CTD (yeast) and to
the terminal GpppN (mammals). c, Imp stimulates
MT substrate binding and N7 methylation of the
cap G, and Pol II O form switches from initiation
to processive elongation.
Shatkin Manley. Nature Struc. Biol. 7838 2000
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Multiple Roles of the 5 Cap During Gene
Expression in Eukaryotes.

• Role in pre-mRNA splicing
• Cap-binding complex (CBC) interacts with m7-G cap
• Cap structure enhances formation of spliced mRNA
• For example, cap promotes efficient
  interaction between U1 snRNP and 5splice site
  (early step in spliceosome assembly)
• How cap and splicing components interact
  currently unknown.
• Role in polyadenylation
• Presence of CBC enhances cleavage rxn of
  polydenylation, but minor effect on poly(A)
  addition rxn.
• Appears to stabilize polyadenylation complex
• Role in mRNA transport
• CBC associates with nascent transcripts (even
  when mRNA spliceosome separate)
• Role of CBC-cap complex non-essential but
  significant for mRNP transport
• 1. CBC forms cap-dependent complex w/
  importin-a, a component of the nuclear import
  machinery
• 2. CBC-mRNA exported to cytoplasm
• 3. Importin-ß causes dissociation of CBC-RNA
  complex in cytoplasm
• 4. In cytoplasm, CBC dissociates from capped
  mRNA and allows binding of eIF-4F, a translation
  initiation complex
• 5. CBC recycles back to nucleus

Red dot 5 mRNA cap CBC Cap-binding
complex Pab1p cytoplasmic poly(A)-binding
protein

• Role in mRNA turnover
• Rates of decapping are primary determinants of
  mRNA half-lives
• Interaction between cap and 3 poly(A) tail
  regulates mRNA turnover (see below)
• In yeast default degradation pathway, decapping
  follows de-adenylation
• Pab1 (cytoplasmic poly(A)-binding protein)
  protects cap from decapping enzyme
• Role in translation initiation
• Cap plays major role in rate-limiting step of
  translation binding of 40S ribosome subunit to
  mRNA
• Occurs via cap-binding eIF-4F complex in
  association with poly(A) tail
• eIF-4F component eIF-4E binds cap
• IRES in some mRNAs bypass requirement for cap in
  translation initiation

Varani. Structure 5855 1997
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Polyadenylation

• CPSF binds to upstream AAUAAA polyadenylation
  signal in RNA transcript
• CstF interacts w/ a downstream GU- or U-rich
  seq., w/ bound CPSF
• Physical links between polyadenylation
  transcription machinery
• CTD of pol II required for efficient
  capping, splicing, 3 end processing
• CPSF CstF associated w/ CTD ? possible
  that poly(A) site recog-
• nition by cleavage factors prerequisite
  for efficient transcrip. term.
• CPSF co-purifies with TFIID ? association of
  pol II w/ cleavage factors
• must occur very early in transcription
  3 subunits of CPSF are part of
• TFIID and are transferred to pol II
  after transcription initiation
• Pol II stimulates RNA cleavage, even in
  absence of transcription
• Nuclease component not yet identified
• Alternatively, CTD may recruit cleavage
  factors into an active complex
• CBC also physically contacts polyadenylation
  machinery (via assoc. w/
• CTD) and participates in cleavage
• All these interactions may stabilize
  polyadenylation complex
• Transcription termination dependent on a
  functional poly(A) signal

Both PAP CPSF involved in poly(A) addition
CPSF cleavage polyadenylation specificity
factor CstF cleavage stimulation factor CF
cleavage factors PAP poly(A) polymerase PAB
Poly(A) binding protein
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Yeast mammalian polyadenylation machineries
Known protein components of the yeast (top) and
mammalian (bottom) complexes are shown. CstF
(CF1A in yeast) subunits are shown in light blue,
CPSF (PFI, or a subcomplex, CF II, in yeast) in
red, and PAP in yellow. Yeast Pcf11 and Fip1,
which lack known mammalian homologs, are denoted
with purple letters. Symplekin, which binds
CstF-64 and shares similarity with the yCPSF
component Pta1, is indicated in a darker blue
than the CstF subunits because it is associated
with a CstFCPSF complex but is not a stable
component of either factor. Other similarities
are as follows CstF-77 Rna14, CstF-64 Rna15,
CPSF-160 Cft1, CPSF-100 Cft2, CPSF-73
Brr5/Ysh1, and CPSF-30 Yth1. Mammalian CFI and
yeast Hrp1, both otherwise unrelated RNA binding
proteins, are colored light and dark green,
respectively. The unpurified mammalian CFII is
gray. RNA signal sequences are boxed. Note that
the binding specificities of the yeast CPSFand
CstF complexes are uncertain, and assignments are
based on predictions from the mammalian system.
The arrow indicates the site of endonucleolytic
cleavage.
Shatkin Manley. Nature Struc. Biol. 7838 2000
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mRNA capping, splicing, 3 end formation, and
transcription all closely linked and
functionally interconnected

• 5 Capping enhances both splicing and
  polyadenylation
• Polyadenylation strongly affected by splicing
• Direct association of pol II complex with mRNA
  processing components during
• inititiation, elongation, and termination
• Many RNA processing factors associate with CTD of
  pol II
• CTD heptad aa seq. repeated 26 times in yeast,
  52 times in mammals
• Ser 2 and Ser 5 of CTD undergo
  phosphorylation
• Unphosphorylated CTD correlated with
  transcription initiation
• Conversion to elongation associated with CTD
  phorphorylation by kinases,
• including TFIIH and elongation factor
  P-TEFb
• Phosphorylation of Ser 5 occurs mainly at early
  stages of elongation Ser 2
• phosphorylation occurs later in elongation
  (see Komarnitsky et al. below)
• Many (or all?) polyadenylation factors associate
  w/ CTD
• 3 subunits of CPSF are components of TFIID,
  and transferred to pol II at time
• of transition to elongtion
• CTD stimulates cleavage/polyadenylation activity
  

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Different phosphorylated forms of RNA pol II and
associated mRNA processing factors during
transcription
Guanylyl transferase
(Guanylyl transferase)
Triphosphatase
Methylase
Pol II
Fig. 2. Capping enz. and cap methyltransferase
show different patterns of association during
transcription. The methyltransferase
stays assoc. w/ elongating pol than
either the triphosphatase or capping enz.
(Pol II)
Relative quantitation of PCR rxns.
ChIP studies in yeast Formaldehyde
cross-linking Lyse cells, shear chromatin
IP w/ appropriate antibody Reverse
cross-links PCR analysis of region of interest
Fig. 1. Capping enz. is localized to promoter
regions.
internal bkgrd. control
Komarnitsky et al. Genes Devel. 142452 2000
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Guanylyl transferase
Yeast CFIB polyaden. factor
Pol II
CTD YSPTSPS (2) (5)
Fig. 4. mRNA cleavage factor IB (Hrp1)
cross-links throughout gene.
Fig. 5. Phosphorylation of CTD ser 5 is
localized to promoters.
Komarnitsky et al. Genes Devel. 142452 2000
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Ab to unphosphoryl. CTD
Kinase of TFIIH phosphorylates ser 5 of CTD
Fig. 7. Ser 2 phosphorylation of the CTD is
localized to coding regions of the gene.
Fig. 6. The TFIIH kinase (Kin28) is required for
CTD ser 5 phosphorylation and capping enz.
recruitment to promoters in vivo.
Komarnitsky et al. Genes Devel. 142452 2000
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Model 1. Unphosphorylated pol II assembles with
PIC at promoter CTD can interact with
transcription factors/complexes. 2. CTD
phophorylated at ser 5 by TFIIH kinase subunit
(KIN28) this may dissociate transcription
factors. 3. CTD phophorylation is signal for
binding of capping enzs. (triphosphatase
guanylyltransferase). 4. After elongation lt200
nucleotides, CTD dephosphorylated at ser 5. 5.
Capping methyltransferase slowly dissociates from
pol II. 6. At least one polyadenylation factor
(Hrp1) remains associated with pol II elongation
complex. 7. As elongation proceeds, level of
CTD phosphorylation at ser 2 increases (kinase
unknown) could act as a recognition site
for other factors in elongation, termination, 3
end processing.
Komarnitsky et al. Genes Devel. 142452 2000