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