The two distinct proteins encoded by the CDKN2A locus are specified by translating the common second exon in alternative reading frames. The product of the α transcript, p16 INK4a , is a recognized tumour suppressor that induces a G 1 cell cycle arrest by inhibiting the phosphorylation of the retinoblastoma protein by the cyclin-dependent kinases, CDK4 and CDK6. In contrast, the product of the human CDKN2A β transcript, p14 ARF , activates a p53 response manifest in elevated levels of MDM2 and p21 CIP1 and cell cycle arrest in both G 1 and G 2 /M. As a consequence, p14 ARFinduced cell cycle arrest is p53 dependent and can be abrogated by the co-expression of human papilloma virus E6 protein. p14 ARF acts by binding directly to MDM2, resulting in the stabilization of both p53 and MDM2. Conversely, p53 negatively regulates p14 ARF expression and there is an inverse correlation between p14 ARF expression and p53 function in human tumour cell lines. However, p14 ARF expression is not involved in the response to DNA damage. These results place p14 ARF in an independent pathway upstream of p53 and imply that CDKN2A encodes two proteins that are involved in tumour suppression.

The CDKN2A tumour suppressor locus encodes two distinct proteins, p16 INK4a and p14 ARF , both of which have been implicated in replicative senescence, the state of permanent growth arrest provoked in somatic cells by aberrant proliferative signals or by cumulative population doublings in culture. Here we describe primary ®broblasts from a member of a melanomaprone family who is homozygous for an intragenic deletion in CDKN2A. Analyses of the resultant gene products imply that the cells are p16 INK4a de®cient but express physiologically relevant levels of a frameshift protein that retains the known functions of p14 ARF . Although they have a ®nite lifespan, the cells are resistant to arrest by oncogenic RAS. Indeed, ectopic expression of RAS and telomerase (hTERT) results in outgrowth of anchorage-independent colonies that have essentially diploid karyotypes and functional p53. We ®nd that in human ®broblasts, ARF is not induced demonstrably by RAS, pointing to signi®cant differences between the proliferative barriers implemented by the CDKN2A locus in different cell types or species.

A number of small RNA molecules that are high affinity ligands for the 46-kDa form of human 2-5 oligoadenylate synthetase have been identified by the SELEX method. Surface plasmon resonance analysis indicates that these RNAs bind to the enzyme with dissociation constants in the nanomolar range. Competition experiments indicate that the binding site for the small RNAs on the 2-5 oligoadenylate synthetase molecule at least partially overlaps that for the synthetic doublestranded RNA, poly(I)⅐poly(C). Several of the RNAs function as potent activators of 2-5 oligoadenylate synthetase in vitro, although there is no correlation between binding affinity and ability to activate. The RNA aptamers having the strongest activation potential appear to have few base-paired regions. This suggests that 2-5 oligoadenylate synthetase, which has previously been believed to be activated only by double-stranded RNA, can also be activated by RNA ligands with little secondary structure. Since 2-5 oligoadenylate synthetase possesses no homology to other known RNA-binding proteins, the development of small specific ligands by SELEX should facilitate studies of RNA-protein interactions and may reveal novel features of the structurefunction relationships involving this enzyme. Double-stranded RNA (dsRNA) 1 binds to and activates a number of interferon-induced proteins, namely the family of 2Ј-5Ј oligoadenylate synthetases (2-5A synthetase) and the protein kinase PKR (reviewed in Refs.

The La (SS-B) autoimmune antigen is an RNA-binding protein that is present in both nucleus and cytoplasm of eukaryotic cells. The spectrum of RNAs that interact with the La antigen includes species which also bind to the interferon-inducible protein kinase PKR. We have investigated whether the La antigen can regulate the activity of PKR and have observed that both the autophosphorylation of the protein kinase that accompanies its activation by dsRNA and the dsRNA-dependent phosphorylation of the alpha subunit of polypeptide chain initiation factor eIF-2 by PKR are inhibited in the presence of recombinant La antigen. This inhibition is partially relieved at higher concentrations of dsRNA. Once activated by dsRNA the protein kinase activity of PKR is insensitive to the La antigen. We have demonstrated by a filter binding assay that La is a dsRNA binding protein. Furthermore, when recombinant La is incubated with a 900 bp synthetic dsRNA or with naturally occurring reovirus dsRNA it converts these substrates to single-stranded forms. We conclude that the La antigen inhibits the dsRNA-dependent activation of PKR by binding and unwinding dsRNA and that it may therefore play a role in the regulation of this protein kinase in interferon-treated or virus-infected cells.