In a BCR/ABL-expressing myeloid precursor cell line, p53 levels were markedly downmodulated. Expression of MDM2, the negative regulator of p53, was upregulated in a tyrosine kinase-dependent manner in growth factor-independent BCR/ABL-expressing cells, and in accelerated phase and blast crisis CML samples. Increased MDM2 expression was associated with enhanced mdm2 mRNA translation, which required the interaction of the La antigen with mdm2 5' UTR. Expression of MDM2 correlated with that of La and was suppressed by La siRNAs and by a dominant negative La mutant, which also enhanced the susceptibility to drug-induced apoptosis of BCR/ABL-transformed cells. By contrast, La overexpression led to increased MDM2 levels and enhanced resistance to apoptosis. Thus, La-dependent activation of mdm2 translation might represent an important molecular mechanism involved in BCR/ABL leukemogenesis.

The mammalian cell nucleus contains structurally stable functional compartments. We show here that one of them, the Cajal body (CB), can be formed de novo. Immobilization on chromatin of both CB structural components, such as coilin, and functional components of the CB, such as the SMN complex, spliceosomal small nuclear ribonucleoproteins (RNPs), small nucleolar RNPs, and small Cajal body-specific RNPs, is sufficient for the formation of a morphologically normal and apparently functional CB. Biogenesis of the CB does not follow a hierarchical assembly pathway and exhibits hallmarks of a self-organizing structure.

By sequence-specific binding to 3' UUU-OH, the La protein shields precursor (pre)-RNAs from 3' end digestion and is required to protect defective pre-transfer RNAs from decay. Although La is comprised of a La motif and an RNA-recognition motif (RRM), a recent structure indicates that the RRM beta-sheet surface is not involved in UUU-OH recognition, raising questions as to its function. Progressively defective suppressor tRNAs in Schizosaccharomyces pombe reveal differential sensitivities to La and Rrp6p, a 3' exonuclease component of pre-tRNA decay. 3' end protection is compromised by mutations to the La motif but not the RRM surface. The most defective pre-tRNAs require a second activity of La, in addition to 3' protection, that requires an intact RRM surface. The two activities of La in tRNA maturation map to its two conserved RNA-binding surfaces and suggest a modular model that has implications for its other ligands.

Termination by RNA polymerase III (Pol III) produces RNAs whose 3 oligo(U) termini are bound by La protein, a chaperone that protects RNAs from 3 exonucleases and promotes their maturation. Multiple reports indicate that yeasts use La-dependent and -independent pathways for tRNA maturation, with defective pre-tRNAs being most sensitive to decay and most dependent on La for maturation and function. The Rpc11p subunit of Pol III shows homology with the zinc ribbon of TFIIS and is known to mediate RNA 3 cleavage and to be important for termination. We used a La-dependent opal suppressor, tRNA Ser UGAM, which suppresses ade6-704 and the accumulation of red pigment, to screen Schizosaccaromyces pombe for rpc11 mutants that increase tRNA-mediated suppression. Analyses of two zinc ribbon mutants indicate that they are deficient in Pol III RNA 3 cleavage activity and produce pre-tRNA Ser UGAM transcripts with elongated 3-oligo(U) tracts that are better substrates for La. A substantial fraction of pre-tRNA Ser UGAM contains too few 3 Us for efficient La binding and appears to decay in wild-type cells but has elongated oligo(U) tracts and matures along the La-dependent pathway in the mutants. The data indicate that Rpc11p limits RNA 3-U length and that this significantly restricts pre-tRNAs to a La-independent pathway of maturation in fission yeast.The 3Ј ends of tRNA and other RNA polymerase III (Pol III)-dependent genes contain dT n termination signals at which Pol III pauses and releases its RNA (6). The role of dT n extends beyond termination, since it provides a means to link Pol III transcripts to La, an abundant and ubiquitous nuclear phosphoprotein that binds these RNAs in a 3Ј-oligo(U) lengthdependent manner and promotes their posttranscriptional processing (27,32,37). Although 3Ј-U length heterogeneity has been well documented for Pol III transcripts (27), relatively little is known about the mechanisms involved and its functional significance. A model system that can be used to alter 3Ј-U length and study its consequences should be helpful in understanding functional connectivity between Pol III termination and posttranscriptional processing.La protein protects pre-tRNAs from 3Ј exonucleases and imposes order on posttranscriptional processing so that 5Ј processing precedes 3Ј processing for many pre-tRNAs (38). Finding the reverse order suggests that different pre-tRNAs use the La-independent and -dependent pathways to various degrees (25). While La-homologous protein (Lhp1p) is nonessential in the yeast Saccharomyces cerevisiae (and in Schizosaccharomyces pombe), its deletion causes lethality or growth deficiency in combination with mutations that impair base pairing or modification of certain pre-tRNAs, indicating that defective pre-tRNAs can be salvaged by La (5,7,21,38). Decay of hypomodified pre-tRNA i Met occurs via 3Ј adenylation and exonucleolytic degradation of pre-tRNA i Met in a process called nuclear surveillance (22) but can be rescued by excess Lhp1p (1, 5).