T cell receptor-beta mRNA splicing: regulation of unusual splicing intermediates.
The expression of functional T cell receptor-beta (TCR-beta) transcripts requires the activation of programmed DNA rearrangement events. It is not clear whether other mechanisms dictate TCR-beta mRNA levels during thymic ontogeny. We examined the potential role of RNA splicing as a regulatory mechanism. As a model system, we used an immature T cell clone, SL12.4, that transcribes a fully rearranged TCR-beta gene but essentially lacks mature 1.3-kb TCR-beta transcripts in the cytoplasm. Abundant TCR-beta splicing intermediates accumulate in the nucleus of this cell clone. These splicing intermediates result from inefficient or inhibited excision of four of the five TCR-beta introns; the only intron that is efficiently spliced is the most 5' intron, IVSL. The focal point for the regulation appears to be IVS1C beta 1 and IVS2C beta 1, since unusual splicing intermediates that have cleaved the 5' splice site but not the 3' splice site of these two introns accumulate in vivo. The block in 3' splice site cleavage is of interest since sequence analysis reveals that these two introns possess canonical splice sites. A repressional mechanism involving a labile repressor protein may be responsible for the inhibition of RNA splicing since treatment of SL12.4 cells with the protein synthesis inhibitor cycloheximide reversibly induces a rapid and dramatic accumulation of fully spliced TCR-beta transcripts in the cytoplasm, concomitant with a decline in TCR-beta pre-mRNAs in the nucleus. This inducible system may be useful for future studies analyzing the underlying molecular mechanisms that regulate RNA splicing.
Qian
L
L
Microbiology and Immunology Department, Vollum Institute for Advanced Biomedical Research, Portland, Oregon.
Theodor
L
L
Carter
M
M
Vu
M N
MN
Sasaki
A W
AW
Wilkinson
M F
MF
D000818:Animals; D001483:Base Sequence; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000378:metabolism; D002999:Clone Cells; D016384:Consensus Sequence; D003513:Cycloheximide / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D015342:DNA Probes; D007438:Introns / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics*; D012097:Repressor Proteins / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology*
D015342:DNA Probes; D009841:Oligonucleotides; D012322:RNA Precursors; D012333:RNA, Messenger; D016693:Receptors, Antigen, T-Cell, alpha-beta; D012097:Repressor Proteins; D003513:Cycloheximide