Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is one of the most abundant RNA binding proteins. roles in gene expression by regulating the biogenesis and translation of messengers RNAs the processing of miRNAs affecting transcription and controlling telomere maintenance. The multiple functions of this protein correlate with diverse roles in genetic disease cancer and the replication of viral pathogens. Utilizing a tagged hnRNP A1 deletion library we have shown that this three hnRNP A1 RBDs contribute to the prevalent nuclear distribution of the protein. Our data also indicate that a truncated form of the protein lacking one of the RBDs the RGG-box can regulate splicing of a splicing reporter minigene and down-regulate replication of the HIV-1 virus with efficiency comparable to the wild type protein. This functional hnRNP A1 deletion mutant is similar to a predicted hnRNP A1 isoform which had not been previously experimentally characterized. experimental data indicates that it is responsible for the cooperative binding of the protein to its target RNA [9 10 hnRNP A1 plays a prominent role in viral replication and pathogenesis. Biochemical and functional studies aimed at the characterization of the complex mechanisms regulating the expression of the HIV-1 genome have shown that hnRNP A1 is usually a part of a complex regulatory network that regulates viral mRNA export stability and splicing . The mechanisms that regulate HIV-1 gene expression utilize both cellular and viral factors which interact with several coding exon that is required for the splicing regulation of coding mRNAs [14 15 hnRNP A1 binds a Glycyrrhizic acid short regulatory sequence named exonic splicing silencer 2 (ESS2) to repress splicing of the messenger. A similar regulatory mechanism has been Glycyrrhizic acid also characterized within the second coding exon [16 17 Over-expression of hnRNP A1 in an cellular system down-regulates expression of the transcriptional transactivator Tat which results in a sharp reduction in the transcription of the viral genome and a 100 fold drop in the production of new virions . The understanding of how each structural domain name and sequence motif of hnRNP A1 contribute to the functions of the proteins is usually key in understanding a number of molecular mechanisms and contribute to the development of novel therapies for genetic and viral diseases. We set up to study the Glycyrrhizic acid role that the different domains and functional motifs of this protein play in cellular localization splicing and viral replication. Furthermore we wanted to determine if a minimal truncated isoform of this protein could recapitulate the effect of the full-length protein in HIV-1 splicing and replication. Here we report that additional sequence motifs other than the M9 domain name are required for the proper cellular localization of the protein and that the RGG-box is not essential for the protein functions in alternative splicing. An hnRNP A1 clone lacking the RGG-box and a portion of the PrLD is usually properly Rabbit Polyclonal to SMC1. localized and can functionally substitute for the ubiquitously expressed A1-A isoform. Furthermore analysis of protein and gene sequence databases indicates that an isoform of hnRNP A1 lacking the RGG-box similar to the functional deletion clone we characterized may be expressed in specific human tissues. 2 Materials and methods 2.1 Plasmids and cell transfections The hnRNP A1 Glycyrrhizic acid deletion clones library was obtained by PCR amplification and cloning into the pEGFP-N1 vector (Clontech) of hnRNP A1-A fragments from the pGT7-A1 expression vector (donation from Dr. A. Krainer CSHL). Single hnRNP A1-A domains and subdomains were amplified utilizing primers F1 (agctccgcggtcttcaccctgccgtcatgtctaag) F2 (agctccgcggatgcccgaacagctgaggaagctcttc) F3 (atgcccgcggatgggaagcaattttggaggtggtg) F4 (agctccgcggatggtctccagagaagattctcaaagac) F5 (atgcccgcggatggctagtgcttcatccagccaaag) R1 (atgcggatccccaaatcttctgccactgccatagctac) R2 (atgcggatccccaccgccatagccaccttggtttcg) R3 (atgcggatccccgtaattcccaaaatcattgtagc) R4 (atgcccgcggcccacttcaatttttccatactgttc) R5 (atgcccgcggggcacctggtctttgagaatcttc) R6 (atgcccgcggtccagaaccacttcgacctctttg) R7 (atgcccgcggcctttctgaatgacaatcttatccac) and cloned following the diagram shown in physique S1. HEK-293 cells were maintained at below 80% confluence in D-MEM (Gibco BRL) supplemented with 8% fetal calf serum.