Translation of MMTV Gag requires nuclear events involving splicing motifs in addition to the viral Rem protein and RmRE

Ioana Boeras, Michael Sakalian, John T. West

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Background: Retroviral Gag proteins are encoded in introns and, because of this localization, they are subject to the default pathways of pre-mRNA splicing. Retroviruses regulate splicing and translation through a variety of intertwined mechanisms, including 5'- post-transcriptional control elements, 3'- constitutive transport elements, and viral protein RNA interactions that couple unspliced and singly spliced mRNAs to transport machinery. Sequences within the gag gene termed inhibitory or instability sequences also appear to affect viral mRNA stability and translation, and the action of these sequences can be countered by silent mutation or the presence of RNA interaction proteins like HIV-1 Rev. Here, we explored the requirements for mouse mammary tumor virus (MMTV) Gag expression using a combination of in vivo and in vitro expression systems.Results: We show that MMTV gag alleles are inhibited for translation despite possessing a functional open reading frame (ORF). The block to expression was post-transcriptional and targeted the mRNA but was not a function of mRNA transport or stability. Using bicistronic reporters, we show that inhibition of gag expression imparted a block to both cap-dependent and cap-independent translation onto the mRNA. Direct introduction of in vitro synthesized gag mRNA resulted in translation, implying a nuclear role in inhibition of expression. The inhibition of expression was overcome by intact proviral expression or by flanking gag with splice sites combined with a functional Rem-Rem response element (RmRE) interaction.Conclusions: Expression of MMTV Gag requires nuclear interactions involving the viral Rem protein, its cognate binding target the RmRE, and surprisingly, both a splice donor and acceptor sequence to achieve appropriate signals for translation of the mRNA in the cytoplasm.

Original languageEnglish (US)
Article number8
JournalRetrovirology
Volume9
DOIs
StatePublished - Jan 25 2012

Fingerprint

Mouse mammary tumor virus
Response Elements
Viral Proteins
Protein Biosynthesis
Messenger RNA
gag Genes
gag Gene Products
RNA Precursors
RNA Stability
Viral RNA
Retroviridae
Introns
Open Reading Frames
HIV-1
Carrier Proteins
Cytoplasm
Alleles
RNA
Proteins
In Vitro Techniques

Keywords

  • Betaretrovirus
  • Gag
  • Post-transcriptional regulation
  • Rem

ASJC Scopus subject areas

  • Virology
  • Infectious Diseases

Cite this

Translation of MMTV Gag requires nuclear events involving splicing motifs in addition to the viral Rem protein and RmRE. / Boeras, Ioana; Sakalian, Michael; West, John T.

In: Retrovirology, Vol. 9, 8, 25.01.2012.

Research output: Contribution to journalArticle

@article{12305c87bc34462e8b9f393649337278,
title = "Translation of MMTV Gag requires nuclear events involving splicing motifs in addition to the viral Rem protein and RmRE",
abstract = "Background: Retroviral Gag proteins are encoded in introns and, because of this localization, they are subject to the default pathways of pre-mRNA splicing. Retroviruses regulate splicing and translation through a variety of intertwined mechanisms, including 5'- post-transcriptional control elements, 3'- constitutive transport elements, and viral protein RNA interactions that couple unspliced and singly spliced mRNAs to transport machinery. Sequences within the gag gene termed inhibitory or instability sequences also appear to affect viral mRNA stability and translation, and the action of these sequences can be countered by silent mutation or the presence of RNA interaction proteins like HIV-1 Rev. Here, we explored the requirements for mouse mammary tumor virus (MMTV) Gag expression using a combination of in vivo and in vitro expression systems.Results: We show that MMTV gag alleles are inhibited for translation despite possessing a functional open reading frame (ORF). The block to expression was post-transcriptional and targeted the mRNA but was not a function of mRNA transport or stability. Using bicistronic reporters, we show that inhibition of gag expression imparted a block to both cap-dependent and cap-independent translation onto the mRNA. Direct introduction of in vitro synthesized gag mRNA resulted in translation, implying a nuclear role in inhibition of expression. The inhibition of expression was overcome by intact proviral expression or by flanking gag with splice sites combined with a functional Rem-Rem response element (RmRE) interaction.Conclusions: Expression of MMTV Gag requires nuclear interactions involving the viral Rem protein, its cognate binding target the RmRE, and surprisingly, both a splice donor and acceptor sequence to achieve appropriate signals for translation of the mRNA in the cytoplasm.",
keywords = "Betaretrovirus, Gag, Post-transcriptional regulation, Rem",
author = "Ioana Boeras and Michael Sakalian and West, {John T.}",
year = "2012",
month = "1",
day = "25",
doi = "10.1186/1742-4690-9-8",
language = "English (US)",
volume = "9",
journal = "Retrovirology",
issn = "1742-4690",
publisher = "BioMed Central",

}

TY - JOUR

T1 - Translation of MMTV Gag requires nuclear events involving splicing motifs in addition to the viral Rem protein and RmRE

AU - Boeras, Ioana

AU - Sakalian, Michael

AU - West, John T.

PY - 2012/1/25

Y1 - 2012/1/25

N2 - Background: Retroviral Gag proteins are encoded in introns and, because of this localization, they are subject to the default pathways of pre-mRNA splicing. Retroviruses regulate splicing and translation through a variety of intertwined mechanisms, including 5'- post-transcriptional control elements, 3'- constitutive transport elements, and viral protein RNA interactions that couple unspliced and singly spliced mRNAs to transport machinery. Sequences within the gag gene termed inhibitory or instability sequences also appear to affect viral mRNA stability and translation, and the action of these sequences can be countered by silent mutation or the presence of RNA interaction proteins like HIV-1 Rev. Here, we explored the requirements for mouse mammary tumor virus (MMTV) Gag expression using a combination of in vivo and in vitro expression systems.Results: We show that MMTV gag alleles are inhibited for translation despite possessing a functional open reading frame (ORF). The block to expression was post-transcriptional and targeted the mRNA but was not a function of mRNA transport or stability. Using bicistronic reporters, we show that inhibition of gag expression imparted a block to both cap-dependent and cap-independent translation onto the mRNA. Direct introduction of in vitro synthesized gag mRNA resulted in translation, implying a nuclear role in inhibition of expression. The inhibition of expression was overcome by intact proviral expression or by flanking gag with splice sites combined with a functional Rem-Rem response element (RmRE) interaction.Conclusions: Expression of MMTV Gag requires nuclear interactions involving the viral Rem protein, its cognate binding target the RmRE, and surprisingly, both a splice donor and acceptor sequence to achieve appropriate signals for translation of the mRNA in the cytoplasm.

AB - Background: Retroviral Gag proteins are encoded in introns and, because of this localization, they are subject to the default pathways of pre-mRNA splicing. Retroviruses regulate splicing and translation through a variety of intertwined mechanisms, including 5'- post-transcriptional control elements, 3'- constitutive transport elements, and viral protein RNA interactions that couple unspliced and singly spliced mRNAs to transport machinery. Sequences within the gag gene termed inhibitory or instability sequences also appear to affect viral mRNA stability and translation, and the action of these sequences can be countered by silent mutation or the presence of RNA interaction proteins like HIV-1 Rev. Here, we explored the requirements for mouse mammary tumor virus (MMTV) Gag expression using a combination of in vivo and in vitro expression systems.Results: We show that MMTV gag alleles are inhibited for translation despite possessing a functional open reading frame (ORF). The block to expression was post-transcriptional and targeted the mRNA but was not a function of mRNA transport or stability. Using bicistronic reporters, we show that inhibition of gag expression imparted a block to both cap-dependent and cap-independent translation onto the mRNA. Direct introduction of in vitro synthesized gag mRNA resulted in translation, implying a nuclear role in inhibition of expression. The inhibition of expression was overcome by intact proviral expression or by flanking gag with splice sites combined with a functional Rem-Rem response element (RmRE) interaction.Conclusions: Expression of MMTV Gag requires nuclear interactions involving the viral Rem protein, its cognate binding target the RmRE, and surprisingly, both a splice donor and acceptor sequence to achieve appropriate signals for translation of the mRNA in the cytoplasm.

KW - Betaretrovirus

KW - Gag

KW - Post-transcriptional regulation

KW - Rem

UR - http://www.scopus.com/inward/record.url?scp=84856118529&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84856118529&partnerID=8YFLogxK

U2 - 10.1186/1742-4690-9-8

DO - 10.1186/1742-4690-9-8

M3 - Article

C2 - 22277305

AN - SCOPUS:84856118529

VL - 9

JO - Retrovirology

JF - Retrovirology

SN - 1742-4690

M1 - 8

ER -