Biomedical subjects

P S McCaw

Publications and source records attributed to P S McCaw.

5 recordsLinked to original sources

PubMed1997

SC35-mediated reconstitution of splicing in U2AF-depleted nuclear extract.

Assembly of the mammalian spliceosome is known to proceed in an ordered fashion through several discrete complexes, but the mechanism of this assembly process may not be universal. In an early step, pre-mRNAs are committed to the splicing pathway through association with U1 small nuclear ribonucleoprotein (snRNP) and non-snRNP splicing factors, including U2AF and members of the SR protein family. As a means of studying the steps of spliceosome assembly, we have prepared HeLa nuclear extracts specifically depleted of the splicing factor U2AF. Surprisingly, the SR protein SC35 can functionally substitute for U2AF65 in the reconstitution of pre-mRNA splicing in U2AF-depleted extracts. This reconstitution is substrate-specific and is reminiscent of the SC35-mediated reconstitution of splicing in extracts depleted of U1 snRNP. However, SC35 reconstitution of splicing in U2AF-depleted extracts is dependent on the presence of functional U1 snRNP. These observations suggest that there are at least three distinguishable mechanisms for the binding of U2 snRNP to the pre-mRNA, including U2AF-dependent and -independent pathways.

Cell Nucleus↗

PubMed1997

A minimal spliceosomal complex A recognizes the branch site and polypyrimidine tract.

The association of U2 snRNP with the pre-mRNA branch region is a critical step in the assembly of spliceosomal complexes. We describe an assembly process that reveals both minimal requirements for formation of a U2 snRNP-substrate RNA complex, here designated the Amin complex, and specific interactions with the branch site adenosine. The substrate is a minimal RNA oligonucleotide, containing only a branch sequence and polypyrimidine tract. Interactions at the branch site adenosine and requirements for polypyrimidine tract-binding proteins for the Amin complex are the same as those of authentic prespliceosome complex A. Surprisingly, Amin complex formation does not require U1 snRNP or ATP, suggesting that these factors are not necessary for stable binding of U2 snRNP per se, but rather are necessary for accessibility of components on longer RNA substrates. Furthermore, there is an ATP-dependent activity that releases or destabilizes U2 snRNP from branch sequences. The simplicity of the Amin complex will facilitate a detailed understanding of the assembly of prespliceosomes.

Adenosine Triphosphate↗

PubMed1989

The gene for enhancer binding proteins E12/E47 lies at the t(1;19) breakpoint in acute leukemias.

The gene (E2A) that codes for proteins with the properties of immunoglobulin enhancer binding factors E12/E47 was mapped to chromosome region 19p13.2-p13.3, a site associated with nonrandom translocations in acute lymphoblastic leukemias. The majority of t(1;19)(q23;p13)-carrying leukemias and cell lines studied contained rearrangements of E2A as determined by DNA blot analyses. The rearrangements altered the E2A transcriptional unit, resulting in the synthesis of a transcript larger than the normal-sized E2A mRNAs in one of the cell lines with this translocation. These observations indicate that the gene for a transcription factor is located at the breakpoint of a consistently recurring chromosomal translocation in many acute leukemias and suggest a direct role for alteration of such factors in the pathogenesis of some malignancies.

Child↗

PubMed1989

Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence.

A DNA binding and dimerization motif, with apparent amphipathic helices (the HLH motif), has recently been identified in various proteins, including two that bind to immunoglobulin enhancers (E12 and E47). We show here that various HLH proteins can bind as apparent heterodimers to a single DNA motif and also, albeit usually more weakly, as apparent homodimers. The HLH domain can mediate heterodimer formation between either daughterless, E12, or E47 (Class A) and achaete-scute T3 or MyoD (Class B) to form proteins with high affinity for the kappa E2 site in the immunoglobulin kappa chain enhancer. The achaete-scute T3 and MyoD proteins do not form kappa E2-binding heterodimers together, and no active complex with N-myc was evident. The formation of a heterodimer between the daughterless and achaete-scute T3 products may explain the similar phenotypes of mutants at these two loci and the genetic interactions between them. A role of E12 and E47 in mammalian development, analogous to that of daughterless in Drosophila, is likely.

Animals↗

PubMed1989

A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins.

Two cDNAs were isolated whose dimerized products bind specifically to a DNA sequence, kappa E2, located in the immunoglobulin kappa chain enhancer. Both cDNAs share a region of extensive identity to the Drosophila daughterless gene and obvious similarity to a segment in three myc proteins, MyoD, and members of the Drosophila achaete-scute and twist gene family. The homologous regions have the potential to form two amphipathic helices separated by an intervening loop. Remarkable is the stringent conservation of hydrophobic residues present in both helices. We demonstrate that this new motif plays a crucial role in both dimerization and DNA binding.

Amino Acid Sequence↗