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 a nd -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 d erived from a single gene through alternative splicing and are evolutionari ly conserved nuclear phosphoproteins that interact with double-stranded RNA . Both NFAR-1 and -2 are phosphorylated by PHR, reciprocally co-immunopreci pitate with PKR, and colocalize with the kinase in a diffuse nuclear patter n within the cell. Transfection studies indicate that the NFARs regulate ge ne expression at the level of transcription, probably during the processing of pre-mRNAs, an activity that was increased in fibroblasts lacking PKR. S ubsequent 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 associ ate with both pre-mRNAs and spliced mRNAs in post-transcriptional studies, similar to the known splicing factor ASF/SF-2. Collectively, our data indic ate that the NFARs may facilitate double-stranded RNA-regulated gene expres sion at the. level of post-transcription and possibly contribute to host de fense-related mechanisms in the cell.