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Lecture 16: Processing of viral pre-mRNA
BSCI437Flint 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 pre-
mRNA 5’ ends5’ 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 non-templated polyA tails attached to their 3’ ends.
• PolyA tails also first discovered using viral systems
• PolyA tails are post-transcriptionally added to cellular mRNAs using a series of cis-acting sequences on the mRNA and trans-acting ribonucleoprotein factors
• PolyA tails interact with PolyA-binding 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.
• 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.
Synthesis of 3’ polyA tails: viral mRNAs
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 in-between is lost
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.
Splicing of pre-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