Tat stimulates cotranscriptional capping of HIV mRNA

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Molecular cell, Vol. 10, 585-597
Tat stimulates cotranscriptional capping of HIV
mRNA
Ya-Lin chiu, C.Kiong Ho, Nayanendu Saha, Beate
Schwer, Stewart Shuman, and Tariq M. Rana
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(No Transcript)
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Mammalian capping apparatus consists of Two
components a bifunctional triphosphatase -
guanylyltransferase(Mce1) and a separate cap
methyltransferase(Hcm1)
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HIV-1 Tat is a small RNA Binding protein
requires for efficient transcription of HIV genes
1. Tat binds specifically to a structured RNA
element , TAR, located at the 5-end of the
nascent HIV transcript.
2. Tat contains Two important functional
domains An arginine-rich region that mediates
the binding of Tat to TAR RNA and an activation
domain that mediates interactions with cellular
factors.
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Emerging connections between CTD phosphorylation,
capping, and transcription elongation
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Objective
How capping and methylation of HIV pre mRNAs are
coupled to Pol ll elongation
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Figure 1. Mammalian Capping Enzyme
Is Not a Componentof PICs but Can Associate with
TECs on the HIV-1 Transcription Unit
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Figure 2. HIV-1 mRNA Capping during
Transcription Elongation(A) 32P-CMP-labeled TECs
stalled at different positions on the template
were prepared by stepwise transcription and then
incubated with or without Mce1. After removing
the unbound Mce1 by washing with transcription
buffer, the TECs were incubated with or without
50 µM GTP in reaction mixtures (20 µl) containing
50 mM Tris-HCl, pH 8.0, 5 mM DTT, and 2.5 mM
MgCl2. (B) PAGE analysis of radiolabeled RNAs
isolated from U14 (lanes 14), A22 (lanes 58),
C30 (lanes 912), or U46 (lanes 1316) TECs after
reaction with Mce1 and GTP as indicated above the
lanes. Capped and uncapped species are indicated
by arrows. Lane M contains radiolabeled 18-, 33-,
and 43-mer DNA oligonucleotide size markers. (C
and D) Stalled 32P-CMP-labeled U14 TECs were
incubated with (lane 2) or without Mce1 (lane 1),
washed, and then walked to 30C (lanes 3 and 4).
Labeled RNAs recovered from the U14, and C30 TECs
were analyzed by PAGE.
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Figure 3 Mce1 Binding to Pol II Transcription
Complexes Requires CTD Phosphorylation but Does
Not Require Nascent RNA (A) Stalled A22 TECs were
treated with 10 µg of RNase A for 5 min at 30C
to digest the extruded 5' end of the RNA (A22')
and then walked to position 26G (G26'). (B) PAGE
analysis of radiolabeled RNAs isolated from TECs
at U14 (lanes 1 and 2), at A22 before (lanes 3
and 4) and after (lanes 7 and 8) RNase A
treatment, and after walking from 22A to 26G
without (lanes 5 and 6) or after (lanes 9 and 10)
RNase treatment. The A22 and A22' TECs had been
incubated with or without Mce1 prior to walking
to 26G as indicated above the lanes. (C) A22 and
A22' (RNase treated) TECs that were incubated
with or without Mce1 were isolated and probed by
Western blotting for Pol II and Mce1. PICs were
analyzed in parallel as controls (lanes 1 and
2). (D) PICs were prepared as described in Figure
1. Incubation of the PICs with dATP converted Pol
IIa to Pol IIo by the action of the
TFIIH-associated CTD kinase. (E) PICs that had
been incubated with or without Mce1 were released
by BspE1 digestion and the protein contents were
resolved by 5 SDS-PAGE and probed by Western
blotting for the Pol II largest subunit and Mce1.
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Figure 4. Dissociation of Mce1 from the
Transcription Complex during Elongation Is
Stimulated by a Factor in Nuclear Extract (A)
Stalled A22 TECs were incubated for 10 min in a
capping reaction mixture containing 20 pmol of
Mce1, 50 µM GTP, 50 mM Tris-HCl, pH 8.0, 5 mM
DTT, and 2.5 mM MgCl2. After washing to remove
Mce1 and GTP, the TECs were incubated with or
without nuclear extract (NE 20 µl). After
removing the unbound proteins by washing with
transcription buffer, all four NTPs were added to
chase the TECs to the 168 position. (B) PAGE
analysis of radiolabeled RNAs isolated from the
A22 TECs and TECs chased to the end of the
template after exposure to Mce1 and nuclear
extract as indicated. (C) The presence of Pol II
and Mce1 in the A22 and chased TECs was probed by
Western blotting. PICs were analyzed in parallel
as controls.
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Figure 5. Hcm1 Associates with Transcription
Elongation Complexes and Methylates the 5' Cap of
Nascent RNAs (A) Stalled A22 TECs were 5' 32P-cap
labeled by Mce1, washed, and then incubated with
Hcm1. After washing to remove unbound Hcm1, TECs
were walked to distal template positions,
released from the beads, and then incubated in
methylation reaction mixtures containing 50 mM
Tris-HCl (pH 8.0), 5 mM DTT, and 50 µM
AdoMet. (B) Radiolabeled RNAs isolated from the
stalled TECs were analyzed by PAGE. (C) The cap
structures of the RNAs isolated from the
indicated TECs were analyzed by S1 nuclease
digestion followed by polyethyleneimine-cellulose
TLC (lanes 59). The chromatographic origin (lane
2) and the positions of    -32PGTP (lane 1)
and 32P-labeled cap dinucleotide GpppG (lane 3)
or m7GpppG (lane 4) markers prepared by in vitro
guanylylation and methylation of isolated 17-mer
RNA using Mce1 and Hcm1 are indicated on the
left. (D) The extent of cap methylation of the
nascent cap-labeled RNAs by the indicated TECs
was quantitated by scanning the TLC plate with a
phosphorimager. (E) PICs (lanes 1 and 2) and
stalled U14 TECs (lane 3) were incubated with 20
pmol of HA-tagged Hcm1 for 10 min at 30C. The
U14 TECs were walked to A22 (lane 4) or U46 (lane
5). Nuclear extract was added to TECs stalled at
U46, washed to remove unbound proteins, and the
TECs were chased to 168 (lane 6). The presence
of Pol II and HA-Hcm1 in the PICs and TECs was
probed by Western blotting
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Figure 6. Tat Stimulates Cotranscriptional
Capping by Mce1 (A) Nascent RNAs were labeled
internally with 2P-CMP during stepwise
transcription. TECs stalled at U14 were incubated
with Mce1. After washing to removing unbound
Mce1, TECs were chased for 90s in the absence or
presence of the Tat proteins (10 pmol) as
indicated above the lanes in (B). RNAs isolated
from TECs were incubated with Hcm1 as described
in Figure 5. m7G-capped RNAs were isolated by
eIF4E affinity chromatography. (B) PAGE analysis
of the input RNA (I), the RNAs bound by the
GST-eIF4E fusion proteins (B), and the uncapped
RNAs in the supernatant phase (S). To evaluate
the specificity and efficiency of the capture
assay, G-capped transcripts were isolated from
stalled A22 TECs treated with Mce1 and analyzed
by eIF4E affinity chromatography (lanes 3 and 4)
in parallel with m7G-capped transcripts isolated
from stalled A22 TECs treated with Mce1 and Hcm1
plus AdoMet (lanes 5 and 6). Other samples are as
follows transcripts isolated from stalled U14
and A22 TECs (lanes 1 and 2) transcripts
isolated from U14 TECs after a chase with 4 NTPs
in the absence (lanes 79) or presence of Tat
(lanes 1012), Tat    2/36 (lanes 1315), or
Tat48     (lanes 1618). (C) The percent of the
input RNA sample that bound to eIF4E was
quantitated by scanning the gel in (B) with a
phosphorimager.
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(No Transcript)
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Discussion

• Freeze-frame analysis of cap formation and
  capping enzyme recruitment during transcription
  by human Pol ll
• Cap formation during continuous Pol ll elongation
• Tat stimulates cotranstcriptional capping of HIV
  mRNA