Charles C. Query

RNA recognition: towards identifying determinants of specificity

Members of a family of proteins containing a conserved approximately 80-amino acid RNA recognition motif (RRM) bind specifically to a wide variety of RNA molecules. Structural studies, in combination with sequence alignments, indicate the structural context of both conserved and non-conserved elements in the motif. These analyses suggest that all RRM proteins share a common fold and a similar protein-RNA interface, and that non-conserved residues contribute additional contacts for sequence-specific RNA recognition.

A common RNA recognition motif identified within a defined U1 RNA binding domain of the 70K U1 snRNP protein

We have defined the RNA binding domain of the 70K protein component of the U1 small nuclear ribonucleoprotein to a region of 111 amino acids. This domain encompasses an octamer sequence that has been observed in other proteins associated with RNA, but has not previously been shown to bind directly to a specific RNA sequence. Within the U1 RNA binding domain, an 80 amino acid consensus sequence that is conserved in many presumed RNA binding proteins was discerned. This sequence pattern appears to represent an RNA recognition motif (RRM) characteristic of a distinct family of proteins. By site-directed mutagenesis, we determined that the 70K protein consists of 437 amino acids (52 kd), and found that its aberrant electrophoretic migration is due to a carboxy-terminal charged domain structurally similar to two Drosophila proteins (su(wa) and tra) that may regulate alternative pre-messenger RNA splicing.

A human autoimmune protein associated with U1 RNA contains a region of homology that is cross-reactive with retroviral p30gag antigen

cDNA encoding a 70 kd protein (70K) associated with U1 small nuclear ribonucleoprotein (snRNP) was cloned from a human brain-stem library using autoantibodies from patients with connective tissue disease. The cDNA-derived amino acid sequence contains 23 residues homologous to a region of murine leukemia virus group-specific antigen p30gag. The homology residues in an antigenic portion of the 70K protein and is defined by a core consensus sequence, ETPEEREERRR, that occurs as a tandem repeat in p30gag of most mammalian type C retroviruses. Anti-p30gag antibodies recognized a recombinant 70K-LacZ fusion protein as well as U1 snRNPs. Using synthetic peptides as competitors, we demonstrated that the region of homology encompasses the site of immunological cross-reactivity. Thus autoantibodies against U1 snRNPs were elicited by immunization with p30gag. On the basis of these findings, we suggest a role for retroviruses in the initiation of autoimmunity.

Nuclear RNA-binding proteins.

Publisher Summary The control of gene expression involves several steps at which specific sequences in pre-mRNA transcripts, as well as those in small RNA molecules, are recognized by proteins. RNA-binding proteins can be expected to mediate interactions in a variety of cellular processes, including those occurring in the transcription complex, the spliceosome and ribosome. The members of one family of nuclear proteins that bind to RNA contain a specific RNA recognition motif (RRM). The RRM family of proteins functions at several levels in RNA processing and some family members are involved in tissue-specific and developmentally regulated gene expression. This chapter describes the proteins that contain this RRM and discusses the potential involvement of these proteins in the control of gene expression at the level of RNA processing. These proteins are modular in structure and often contain at least two types of interactive surfaces—one or more that interacts specifically with RNA and another that interacts with other molecules. Studies to date indicate that despite the strong homology among these proteins, they have unique properties of recognition that allow them to distinguish the RNAs of diverse structure.

Expression of autoantibodies to recombinant (U1) RNP-associated 70K antigen in systemic lupus erythematosus.

To determine the specificity of antibodies to the (U1) ribonucleoprotein antigen in systemic lupus erythematosus (SLE), patient sera were tested for binding to a recombinant human 70K antigen. By solid-phase immunoassay, we detected anti-70K reactivity in sera from 31 of 96 patients with systemic lupus erythematosus (SLE), demonstrating that anti-70K antibodies may occur in patients with SLE as well as other clinical diagnoses. In sequential sera from 2 of these patients, we found that anti-70K binding varied dramatically over the course of disease. The changes in anti-70K antibody levels did not correlate with clinical events nor evolving antibody reactivity with the Sm-specific antigens.

The U1 RNA-specific 70K protein: RNA-binding studies and autoimmune cross-reactivity with type-C retroviruses

The predicted amino acid sequence of the 70K protein contained a 23 amino acid region of strong homology to mammalian type-C retroviral p30 gag proteins. The homology resides in a hydrophilic portion of the 70K and p30 gag proteins and is defined by a core sequence, ETPEEREERRR, which occurs as a tandem repeat in most mammalian type-C retrovlruses. Retroviruses have been implicated in the etiology of autoimmunity by the association of xenotropic type-C viruses with NZB autoimmune mice, by the presence of gagreactive antibodies in patients with lupus erythematosus and by the finding of p30gag-llke material in immune complex deposits of nephritis patients. Using antisera against p30 gag, we demonstrated cross-reactivity of the 70K and p30 gag proteins. Antibodies affinity-purified to either the 70K protein or p30 gag reacted with both proteins. Using synthetic peptides, we showed that the region of homology encompasses the site of immunologlcal cross-reactivity. Moreover, sera from autoimmune patients were reactive with this epitope. We will describe a model for the etiology and progression of the anti-U1 RNP response which includes immune presentation of p30 gag protein and subsequent cross-reactivity with the U1 snRNP complex. We suggest that in the proper genetic setting, the production of cross-reacting retroviral antigens may participate in the initiation of the autoimmune response.