No abstract

No abstract

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.

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.The removal of introns from precursors to mRNA molecules (pre-mRNA) is catalyzed by the spliceosome, a dynamic 50S-60S complex composed of small nuclear RNAs (snRNAs) U1, U2, U5, and U4/6, as well as protein components (for review, see references 34, 39, 43, and 46). Such intron excision proceeds by way of two sequential transesterification reactions. The spliceosome assembles de novo on each substrate premRNA, and several distinct intermediates in an assembly pathway can be observed in vitro. The E (early) or commitment complex contains U1 snRNP and non-snRNP protein factors (28,42,58). Complex A is generated by the stable binding of U2 snRNP to the branch region of the pre-mRNA; a larger complex, B, is formed by association of U4/5/6 tri-snRNP with complex A. Complex C follows B after significant rearrangements and contains splicing intermediates (29,30,43).The branch region contains the nucleophile for the first chemical step of splicing, and its recognition is required early in splicing complex assembly. U2 snRNP binds the pre-mRNA, in part, through U2 snRNA ⅐ branch region base pairing (48,69,73), and the first-step nucleophile is selected, in part, by virtue of being bulged from this duplex (51). Early branch site recognition in yeast requires U1 snRNP and a non-snRNP splicing factor, a component of which may be MUD2 (2, 55, 59). In mammals, factors SF3a and SF3b (both of which join 12S U2 to form 17S U2 snRNP), SF1, U2AF 65 , U2AF 35 , U1 snRNP, and members of a family of proteins containing arginine-serine dipeptide repeats (SR proteins; for review, see references 23, 40, and 66) are important for the stable association of U2 snRNP with the pre-mRNA (3,6,7,9,10,33,74). U2AF 65 binds specifically to polypyrimidine tracts (PPTs) in early complexes (24,42,56,70). Another factor, poly(U)-binding factor 2 (PUF-2), which contains two more polypyrimidinebinding proteins, a p54 SR protein (14, 71) and p130, is also important for efficient compl...

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 U2AF 65 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.Pre-mRNA splicing occurs via two sequential transesterification reactions in a 60S complex known as the spliceosome, which assembles on the pre-mRNA substrate in an ordered fashion through several discrete complexes (E, A, B, C; ref. 1). The spliceosome includes the small nuclear ribonucleoprotein (snRNP) particles U1, U2, U4͞6, and U5, as well as associated splicing factors (2-4). Commitment of a pre-mRNA substrate to assembly of the spliceosome involves the ATP-independent formation of the E (early) or commitment complex (5-8). This complex, in the mammalian system, contains U1 snRNP as well as non-snRNP protein factors, including the U2 auxiliary factor, U2AF, and members of the SR protein family (8).SR proteins contain extensive serine͞arginine (SR) repeats and a subset contain RNA recognition motifs; the predominant members of the family are conserved from Drosophila to humans. Many SR proteins are important splicing factors that function in both constitutive and alternative RNA splicing (9). SR proteins containing RNA recognition motifs display modest affinity and sequence specificity in their association with RNA and probably bind cooperatively in association with other factors, including other SR proteins (9). SR family members associate with pre-mRNAs early in spliceosomal assembly (10, 11) and this association may persist through the chemistry of splicing (12). It has been suggested that SR proteins are required for specific transitions during the course of spliceosomal assembly such as the progression from A to B complex (13). The 35-kDa SR protein SC35 has been reported to stimulate E complex formation (8) and has been shown to be associated with a complex formed at the 3Ј end of the intron at early stages in spliceosome assembly (14).Conserved sequence elements at the 5Ј and 3Ј splice sites and in the branch sequence and pyrimidine tract of the pre-mRNA direct formation of the s...