Transcriptional regulation of RANTES expression in T lymphocytes.

We first identified the RANTES chemokine as part of a search for genes expressed by T lymphocytes "late", 3-5 days, after T-cell activation. The kinetics of expression of RANTES and a small number of other genes are unusual and the mechanism of such delayed expression is unknown. In order to uncover a mechanism for such "late" expression, we identified and characterized the RANTES promoter and a novel transcription factor regulating RANTES expression in T lymphocytes. RANTES factor of late activated T lymphocytes (RFLAT)-1 is a member of the Krüppel-like family of transcription factors. Like RANTES, RFLAT-1 expression is "late" after T-cell activation. But, unlike RANTES, regulation of RFLAT-1 expression appears to be translational rather than transcriptional.

Facilitation of group I splicing in vivo: misfolding of the Tetrahymena IVS and the role of ribosomal RNA exons.

Self-splicing of the group I IVS from Tetrahymena thermophila rDNA is limited by the time required for the RNA to reach its active conformation. In vitro, folding is slow because the pre-rRNA becomes kinetically trapped in inactive structures. In vivo, splicing is 50 times more rapid, implying that misfolding of the pre-rRNA is corrected. Exon mutations that inhibit self-splicing 100-fold in vitro were fully rescued when the pre-rRNA containing the IVS was expressed in E. coli. In contrast, IVS mutations that cause misfolding were only partially suppressed at 42 degrees C, and doubled the activation energy of splicing. These results suggest that intracellular folding of the pre-rRNA involves metastable intermediates similar to those observed in vitro. Precursors with natural rRNA exons were more active and less cold-sensitive than those with non-rRNA exons. This shows that the rRNA reduces misfolding of the IVS, thereby facilitating splicing of the pre-rRNA in vivo.

Association of a group I intron with its splice junction in 50S ribosomes: implications for intron toxicity.

The effect of genetic context on splicing of group I introns is not well understood at present. The influence of ribosomal RNA conformation on splicing of rDNA introns in vivo was investigated using a heterologous system in which the Tetrahymena group I intron is inserted into the homologous position of the Escherichia coli 23S rRNA. Mutations that block splicing in E. coli result in accumulation of unspliced 23S rRNA that is assembled into 50S complexes, but not 70S ribosomes. The data indicate that accommodation of the intron structure on the surface of the 50S subunit inhibits interactions with the small ribosomal subunit. Spliced intron RNA also remains noncovalently bound to 50S subunits on sucrose gradients. This interaction appears to be mediated by base pairing between the intron guide sequence and the 23S rRNA, because the fraction of bound intron RNA is reduced by point mutations in the IGS or deletion of the P1 helix. Association of the intron with 50S subunits correlates with slow cell growth. The results suggest that group I introns have the potential to inhibit protein synthesis in prokaryotes by direct interactions with ribosomes.