Selective Translation of Eukaryotic mRNAs: Functional Molecular Analysis of GRSF-1, a Positive Regulator of Influenza Virus Protein Synthesis

Abstract

To understand the regulation of cap-dependent translation initiation mediated by specific 5 untranslated region (UTR) RNA-protein interactions in mammalian cells, we have studied the selective translation of influenza virus mRNAs. Previous work has shown that the host cell mRNA binding protein guanine-rich sequence factor 1 (GRSF-1) bound specifically to conserved viral 5 UTR sequences and stimulated translation of viral 5 UTR-driven mRNAs in vitro. In the present study, we have characterized the functional domains of GRSF-1 and mapped the RNA binding activity of GRSF-1 to RRM 2 (amino acids 194 to 275) with aminoterminal deletion glutathione S-transferase (GST)-GRSF-1 proteins. When these mutants were assayed for functional activity in vitro, deletion of an Ala-rich region (⌬[2-94]) appeared to diminish translational stimulation, while deletion of the Ala-rich region in addition to RRM 1 (⌬[2-194]) resulted in a 4-fold increase in translational activation over wild-type GRSF-1 (an overall 20-fold increase in activity). We have also mapped the GRSF-1 RNA binding site on influenza virus NP and NS1 5 UTRs, which was determined to be the sequence AGGGU. With polysome fractionation and cDNA microarray analysis, we have identified cellular and viral mRNAs containing putative GRSF-1 binding sites that were transcriptionally up-regulated and selectively recruited to polyribosomes following influenza virus infection. Taken together, these studies demonstrate that RRM 2 is critical for GRSF-1 RNA binding and translational activity. Further, our data suggest GRSF-1 functions by selectively recruiting cellular and viral mRNAs containing 5 UTR GRSF-1 binding sites to polyribosomes, which is mediated through interactions with cellular proteins.The ability of cells to respond to extracellular stimuli and intracellular cues, including mitogenic signals, is directly linked to the regulation of mRNA translation initiation. The control of initiation can be regulated by the specific interaction of RNA binding proteins and initiation factors (eIFs) with cisacting elements contained in both the 5Ј and 3Ј untranslated regions (UTRs) of mature mRNAs (reviewed in reference 37). Accordingly, deregulation of protein synthesis is a key mechanism in both malignant transformation and viral replication. Influenza virus infection results in the selective translation of viral mRNAs, while host cell protein synthesis is markedly attenuated (reviewed in reference 38). The subversion of the host cell protein synthetic machinery to produce high levels of influenza virus proteins, which are required for infection and replication, is dependent on conserved sequences present in the 5Ј UTRs of the influenza virus mRNAs (13).Translation initiation relies upon the interactions of transacting factors with both ribosomal RNAs and cis-acting determinants in the mRNA. Influenza virus protein synthesis is a cap-dependent process mediated by highly conserved sequences contained in the 5Ј UTRs of the viral mRNAs (15). As

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“…4B). Interestingly, we also found GRSF-1, a protein recruiting mRNAs to polyribosomes (31) and shown to bind to IRESs (32), highly enriched (SILAC ratio 9) at the wild-type sequence (Fig. 4C A B C of GRSF1 is also enriched specifically with the wild-type sequence in forward and reverse DGCR-8 RNA pull-down.…”

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