Tumor-induced osteomalacia in association with PTEN-negative Cowden syndrome.

Tumor-induced osteomalacia (TIO) is a rare paraneoplastic condition in which phosphaturic mesenchymal tumors (PMTs) secrete high levels of fibroblast growth factor 23 (FGF23) into the circulation. This results in renal phosphate wasting, hypophosphatemia, muscle weakness, bone pain, and pathological fractures. Recent studies suggest that fibronectin-fibroblast growth factor receptor 1 (FN1-FGFR1) translocations may be a driver of tumorigenesis. We present a patient with TIO who also exhibited clinical findings suggestive of Cowden syndrome (CS), a rare autosomal dominant disorder characterized by numerous benign hamartomas, as well as an increased risk for multiple malignancies, such as thyroid cancer. While CS is a clinical diagnosis, most, but not all, harbor a mutation in the tumor suppressor gene PTEN. Genetic testing revealed a somatic FN1-FGFR1 translocation in the FGF23-producing tumor causing TIO; however, a germline PTEN mutation was not identified. To our knowledge, this is the first reported case of concurrent TIO and CS.

Recognition of RNA branch point sequences by the KH domain of splicing factor 1 (mammalian branch point binding protein) in a splicing factor complex.

Mammalian splicing factor 1 (SF1; also mammalian branch point binding protein [mBBP]; hereafter SF1/mBBP) specifically recognizes the seven-nucleotide branch point sequence (BPS) located at 3' splice sites and participates in the assembly of early spliceosomal complexes. SF1/mBBP utilizes a "maxi-K homology" (maxi-KH) domain for recognition of the single-stranded BPS and requires a cooperative interaction with splicing factor U2AF65 bound to an adjacent polypyrimidine tract (PPT) for high-affinity binding. To investigate how the KH domain of SF1/mBBP recognizes the BPS in conjunction with U2AF and possibly other proteins, we constructed a transcriptional reporter system utilizing human immunodeficiency virus type 1 Tat fusion proteins and examined the RNA-binding specificity of the complex using KH domain and RNA-binding site mutants. We first established that SF1/mBBP and U2AF cooperatively assemble in our reporter system at RNA sites composed of the BPS, PPT, and AG dinucleotide found at 3' splice sites, with endogenous proteins assembled along with the Tat fusions. We next found that the activities of the Tat fusion proteins on different BPS variants correlated well with the known splicing efficiencies of the variants, supporting a model in which the SF1/mBBP-BPS interaction helps determine splicing efficiency prior to the U2 snRNP-BPS interaction. Finally, the likely RNA-binding surface of the maxi-KH domain was identified by mutagenesis and appears similar to that used by "simple" KH domains, involving residues from two putative alpha helices, a highly conserved loop, and parts of a beta sheet. Using a homology model constructed from the cocrystal structure of a Nova KH domain-RNA complex (Lewis et al., Cell 100:323-332, 2000), we propose a plausible arrangement for SF1/mBBP-U2AF complexes assembled at 3' splice sites.

The structure of an RNA dodecamer shows how tandem U-U base pairs increase the range of stable RNA structures and the diversity of recognition sites.

BACKGROUND: Non-canonical base pairs are fundamental building blocks of RNA structures. They can adopt geometries quite different from those of canonical base pairs and are common in RNA molecules that do not transfer sequence information. Tandem U-U base pairs occur frequently, and can stabilize duplex formation despite the fact that a single U-U base pair is destabilizing. RESULTS: We determined the crystal structure of the RNA dodecamer GGCGCUUGCGUC at 2.4 A resolution. The molecule forms a duplex containing tandem U-U base pairs, which introduce an overall bend of 11-12 degrees in the duplex resulting from conformational changes at each interface between the tandem U-U base pairs and a flanking duplex sequence. The formation of the U-U base pairs cause small changes in several backbone torsion angles; base stacking is preserved and two hydrogen bonds are formed per base pair, explaining the stability of the structure. CONCLUSIONS: Tandem U-U base pairs can produce stable structures not accessible to normal A-form RNA, which may allow the formation of specific interfaces for RNA-RNA or RNA-protein recognition. These base-pairs show an unusual pattern of hydrogen-bond donors and acceptors in the major and minor grooves, which could also act as a recognition site.

Changes in antiepileptic drug prescribing patterns in large institutions: preliminary results of a five-year experience.

Routine antiepileptic prescriptions were analyzed for stable cohorts from two large, state-run, Accreditation Council-certified ICFs/MR for the time period October 1, 1987, to April 1, 1992. Results indicated marked decrease in numbers of individuals receiving (a) more than two antiepileptic drugs concurrently and (b) barbiturates. Retrospective analyses revealed that (a) over 90% of a group undergoing barbiturate taper maintained the same or improved seizure control and (b) some adults undergoing barbiturate tapers improved in target behaviors. Findings indicate the feasibility of changing antiepileptic regimens utilizing individual case evaluation and treatment and suggest the importance of considering possible psychological and behavioral disturbances associated with barbiturate therapy for adults and children.

A high affinity binding site for the HIV-1 nucleocapsid protein.

The nucleocapsid protein (NC) of HIV-1 is a small zinc finger protein that contributes to multiple steps of the viral life cycle, including the proper encapsidation of HIV RNA. This is accomplished through an interaction between NC and a region at the 5'-end of the RNA, defined as the Psi element. However, the specificity of NC for Psi or for RNA in general is not well understood. To study this problem, we used SELEX to identify high affinity RNA ligands that bind to NC. A 'winner' molecule (SelPsi), as well as a subregion of Psi RNA, were further characterized to understand the interaction between NC and SelPsi and its relationship to the interaction between NC and Psi. The comparison makes predictions about the sequence and structure of a high affinity binding site within the HIV-1 Psi element.

A cooperative interaction between U2AF65 and mBBP/SF1 facilitates branchpoint region recognition.

During the early events of pre-mRNA splicing, intronic cis-acting sequences are recognized and interact through a network of RNA-RNA, RNA-protein, and protein-protein contacts. Recently, we identified a branchpoint sequence binding protein in yeast (BBP). The mammalian ortholog (mBBP/SF1) also binds specifically to branchpoint sequences and interacts with the well studied mammalian splicing factor U2AF65, which binds to the adjacent polypyrimidine (PY) tract. In this paper we demonstrate that the mBBP/SF1-U2AF65 interaction promotes cooperative binding to a branchpoint sequence-polypyrimidine tract-containing RNA, and we suggest that this cooperative RNA binding contributes to initial recognition of the branchpoint sequence (BPS) during pre-mRNA splicing. We also demonstrate the essential nature of the third RBD of U2AF65 for the interaction between the two proteins, both in the presence and absence of RNA.

The KH domain of the branchpoint sequence binding protein determines specificity for the pre-mRNA branchpoint sequence.

The yeast and mammalian branchpoint sequence binding proteins (BBP and mBBP/SF1) contain both KH domain and Zn knuckle RNA-binding motifs. The single KH domain of these proteins is sufficient for specific recognition of the pre-mRNA branchpoint sequence (BPS). However, an interaction is only apparent if one or more accessory modules are present to increase binding affinity. The Zn knuckles of BBP/mBBP can be replaced by an RNA-binding peptide derived from the HIV-1 nucleocapsid protein or by an arginine-serine (RS)7 peptide, without loss of specificity. Only the seven-nucleotide branchpoint sequence and two nucleotides to either side are necessary for RNA binding to the chimeric proteins. Therefore, we propose that all three of these accessory RNA-binding modules bind the phosphate backbone, whereas the KH domain interacts specifically with the bases of the BPS. Proteins and protein complexes with multiple RNA-binding motifs are frequent, suggesting that an intimate collaboration between two or more motifs will be a general theme in RNA-protein interactions.

Crystal structure of a model branchpoint-U2 snRNA duplex containing bulged adenosines.

Bulged nucleotides play a variety of important roles in RNA structure and function, frequently forming tertiary interactions and sometimes even participating in RNA catalysis. In pre-mRNA splicing, the U2 snRNA base pairs with the intron branchpoint sequence (BPS) to form a short RNA duplex that contains a bulged adenosine that ultimately serves as the nucleophile that attacks the 5' splice site. We have determined a 2.18-A resolution crystal structure of a self-complementary RNA designed to mimic the highly conserved yeast (Saccharomyces cerevisiae) branchpoint sequence (5'-UACUAACGUAGUA with the BPS italicized and the branchsite adenosine underlined) base paired with its complementary sequence from U2 snRNA. The structure shows a nearly ideal A-form helix from which two unpaired adenosines flip out. Although the adenosine adjacent to the branchsite adenosine is the one bulged out in the structure described here, either of these adenosines can serve as the nucleophile in mammalian but not in yeast pre-mRNA splicing. In addition, the packing of the bulged RNA helices within the crystal reveals a novel RNA tertiary interaction in which three RNA helices interact through bulged adenosines in the absence of any divalent metal ions.

The splicing factor BBP interacts specifically with the pre-mRNA branchpoint sequence UACUAAC.

The yeast splicing factor BBP (branchpoint bridging protein) interacts directly with pre-mRNA at or very near the highly conserved branchpoint sequence UACUAAC within the commitment complex. We also show that the recombinant protein recognizes the UACUAAC sequence. Therefore, BBP is also an acronym for branchpoint binding protein. The mammalian splicing factor SF1 is a BBP ortholog (mBBP) and an E complex component, and also has branchpoint sequence specificity. The relative conservation of this region in yeast and mammals correlates well with the RNA-binding differences between BBP and mBBP, suggesting that BBP contributes to branchpoint sequence definition in both systems.