Characterization of two evolutionarily conserved, alternatively spliced nuclear phosphoproteins, NFAR-1 and -2, that function in mRNA processing and interact with the double-stranded RNA-dependent protein kinase, PKR.
We report here the isolation and characterization of two proteins, NFAR-1 and -2, which were isolated through their ability to interact with the dsRNA-dependent protein kinase, PKR. The NFAR proteins, of 90 and 110 kDa, are derived from a single gene through alternative splicing and are evolutionarily conserved nuclear phosphoproteins that interact with double-stranded RNA. Both NFAR-1 and -2 are phosphorylated by PKR, reciprocally co-immunoprecipitate with PKR, and colocalize with the kinase in a diffuse nuclear pattern within the cell. Transfection studies indicate that the NFARs regulate gene expression at the level of transcription, probably during the processing of pre-mRNAs, an activity that was increased in fibroblasts lacking PKR. Subsequent functional analyses indicated that amino acids important for NFAR's activity were localized to the C terminus of the protein, a region that was found to specifically interact with FUS and SMN, proteins also known as regulators of RNA processing. Accordingly, both NFARs were found to associate with both pre-mRNAs and spliced mRNAs in post-transcriptional studies, similar to the known splicing factor ASF/SF-2. Collectively, our data indicate that the NFARs may facilitate double-stranded RNA-regulated gene expression at the level of post-transcription and possibly contribute to host defense-related mechanisms in the cell.
Saunders
L R
LR
Department of Microbiology and Immunology and Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, Miami, Florida 33136, USA.
Perkins
D J
DJ
Balachandran
S
S
Michaels
R
R
Ford
R
R
Mayeda
A
A
Barber
G N
GN
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D017931:DNA Primers; D018076:DNA, Complementary; D019143:Evolution, Molecular; D008969:Molecular Sequence Data; D050779:Nuclear Factor 90 Proteins; D010750:Phosphoproteins; D011233:Precipitin Tests; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D012323:RNA Processing, Post-Transcriptional / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D019892:eIF-2 Kinase / Q000378:metabolism*
D017931:DNA Primers; D018076:DNA, Complementary; C429643:ILF3 protein, human; D050779:Nuclear Factor 90 Proteins; D010750:Phosphoproteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D019892:eIF-2 Kinase