Transcript Lecture 15: Processing of viral pre-mRNA

Lecture 16: Processing of
viral pre-mRNA
BSCI437
Flint et al., Chapter 10
GENERAL OVERVIEW
• Viral mRNAs are translated by cellular protein synthetic
apparatus
• They must conform to the requirements of host cell
translation system
• A series of covalent modifications allow this to occur:
RNA processing
• After processing, mRNAs are translated in the cytoplasm
• For viral mRNAs produced in the nucleus, they must be
exported to the cytoplasm
• Once in the cytoplasm, gene expression is a balance
between the translatability of an mRNA and its stability.
• Viral mRNAs have evolved to be very stable.
GENERAL OVERVIEW (Fig. 10.1)
COVALENT MODIFICATIONS DURING
VIRAL PRE-mRNA PROCESSING
• Pre-mRNA modification of cellular mRNAs
is performed in the nucleus
– Addition of 5’ 7Methyl-Gppp caps
– Addition of 3’ poly-adenylated tails
– Splicing
– RNA editing (in some cases. Not discussed in
this lecture).
Capping of
cellular premRNA 5’ ends
5’ 7MeGppp caps
discovered by Shatkin
using Reovirus
Cellular mRNAs capped
cotranscriptionally in the
nucleus by action of 5
enzymes
Functions of the cap:
– Protect 5’ end from
exonucolytic attack
– Interact with translation
initiation apparatus
– Mark mRNAs as “self”
Capping of viral pre-mRNA 5’
ends
• Synthesized by host cell enzymes. e.g.
Retroviruses, Adenoviruses
• Synthesized by viral enzymes, e.g. Poxviruses,
Reoviruses
• Cap snatching: virus steal caps from host
mRNAs. e.g. Influenza
• Note: many RNA viruses have evolved around
requirement for cap.
– Proteins covalently bound to 5’ end substitute for
caps. e.g. Picornaviruses
– Translation initiated internally on mRNA at IRES
elements. e.g. Hepatitis C virus.
Synthesis of 3’ polyA
tails: cellular mRNAs.
• All cellular mRNAs have nontemplated polyA tails attached to
their 3’ ends.
• PolyA tails also first discovered
using viral systems
• PolyA tails are posttranscriptionally added to cellular
mRNAs using a series of cisacting sequences on the mRNA
and trans-acting ribonucleoprotein
factors
• PolyA tails interact with PolyAbinding protein: important for
translation.
(Fig. 10.3)
Synthesis of 3’ polyA tails: viral mRNAs
• Viral mRNAs can be polyadenylated by
host or viral enzymes
• By Host enzymes:
– Occurs like host mRNAs.
– Post-transcriptionally
– Examples: retroviruses, herpesviruses,
adenoviruses.
Synthesis of 3’ polyA tails: viral mRNAs
• By Viral enzymes
• Can occur co-transcriptionally:
– Copying of a long polyU stretch in template RNA:
picornaviruses, M virus of yeast
– Reiteritive copying of short U stretches in template
RNA: Ortho- and Paramyxoviruses
• Can occur post-transcriptionally
– Example: poxviruses
• Note: many viruses have dispensed with polyA tails
altogether. Rather, they trick polyA-binding protein to
interact with complex 3’ mRNA structures.
Splicing of pre-mRNA
Background
• hnRNA: heterogeneous nuclear RNA
– Larger than mRNA
– Has same 5’ and 3’ UTRs as mRNA
– Conclusion: both sides are preserved in
mRNA but somehow information inbetween is lost
Splicing of pre-mRNA
Sharp and Roberts (1993 Nobel Prize).
• The Adenovirus late major mRNA
• Contains sequence derived from 4 different
blocks of genomic sequence
• Precursor late major RNA has the 4 sequence
blocks + all sequence in between.
• Conclusion: in between sequences are “spliced
out” of the pre-mRNA to make the mature
mRNA.
Discovery of splicing.
R-looping: hybridize mRNA with DNA: note that DNA sequences looped
out.
Compare sequences of cDNA versus gDNA with in situ-hybridization/EM
staining.
Splicing: evolutionary implications
• Exons contain protein coding
information
• Shuffling of exons can be used to
create new functional arrangements
• Reflected in modular arrangement of
many proteins.
• Introns facilitate transfer of genetic
information between cellular and viral
genomes
Constitutive vs. Alternative splicing
• Constitutive splicing:
– Every intron is spliced out; Every exon is spliced in
• Alternative splicing:
– All introns spliced out; Only selected exons spliced in
– Result: mRNAs having different coding information derived
from a single gene
•(Fig. 10.8)
Alternative splicing and viruses
• Allows expansion of the limited
coding capacity of viral genomes
• Can be employed to temporally
regulate viral gene expression
• Can control balance in the production
of different regulatory units.
• Can control balance in production
between spliced and unspliced RNAs.
Alternative splicing and viruses
• Spliced RNAs: mRNAs encoding 3’ information. E.g. splicing
of retroviral mRNAs produces mRNAs endoding env gene
• Unspliced RNAs:
– Can encode 5’ genes, e.g. gag-pol of retroviruses Can be used as
‘genomes’ for packaging inside of nascent viral particles (e.g.
retroviruses)
(Fig. 10.11)
POSTTRANSCRIPTIONAL
REGULATION BY VIRAL PROTEINS
• In general: the presence of an mRNA is not
equivalent to the presence of its encoded
protein.
• The extent of translation of an mRNA can be
regulated post-transcriptionally through:
• Regulation of initiation
• Regulation of mRNA stability
• Viral proteins can regulate translation of either
– Viral mRNAs, or
– Cellular mRNAs
Temporal control of gene
expression
Regulation of alternative polyadenylation
• Polyadenylation is required to
translate most mRNAs
• e.g.: Bovine papillomavirus late
mRNA
–Always present
–But, only polyadenylated (and therefore
expressed) late in life cycle.
Temporal control of gene expression
Regulation of splicing
• Control of alternative
splicing is a way to
regulate gene expression
• e.g. Influenza A M1
mRNA (Fig. 10.19)
– Early: Splicosome
recognizes M3 splice
site…makes M3 mRNA
– Late: Viral P proteins
recruit cellular SR protein,
directing splisosome to M3
splice site to make M2
mRNA.
Inhibition of cellular mRNA
production by viral proteins
General notion:
• In the battle between viruses and host cell,
viruses can gain an upper hand by
shutting down cellular functions.
• One approach is to inhibit production of
translation competent cellular mRNAs
Inhibition of polyadenylation
and splicing
• Influenza NS1
protein inhibits
both
polyadenylation
and splicing of
cellular mRNAs
resulting in
preferential
translation of
viral mRNAs
Inhibition of polyadenylation
and splicing
• HSV ICP27
protein
mislocalizes
splicosome
components,
resulting in
inhibition of host
pre-mRNA
splicing.
Regulation of mRNA stability by
a viral protein
• Protein expression = (rate of translation
initiation) x (mRNA half-life)
• The more stable an mRNA is, the more
protein can be synthesized from it
• Many viruses encode proteins that
preferentially destabilize cellular mRNAs
• e.g. virion host shutoff protein (Vhs) of
Herpes simplex encodes an RNase H that
degrades mRNAs