Gene rearrangement during the ontogeny of T-and B-cells generates an enormous repertoire of T-cell receptor (TCR) and immunoglobulin (Ig) genes. Because of the error-prone nature of this rearrangement process, two-thirds of rearranged TCR and Ig genes are expected to be out-of-frame and thus contain premature terminations codons (ptcs). We performed sequence analysis of reverse transcriptase-polymerase chain reaction products from fetal and adult thymus and found that newly transcribed TCR-␤ pre-mRNAs (intron-bearing) are frequently derived from ptc-bearing genes but such transcripts rarely accumulate as mature (fully spliced) TCR-␤ transcripts. Transfection studies in the SL12.4 T-cell line showed that the presence of a ptc in any of several TCR-␤ exons triggered a decrease in mRNA levels. Ptc-bearing TCR-␤ transcripts were selectively depressed in levels in a cell clone that contained both an in-frame and an out-of-frame gene, thus demonstrating the allelic specificity of this down-regulatory response. Protein synthesis inhibitors with different mechanism of action (anisomysin, cycloheximide, emetine, pactamycin, puromycin, and polio virus) all reversed the downregulatory response. Ptc-bearing transcripts were induced within 0.5 h after cycloheximide treatment. The reversal by protein synthesis inhibitors was not restricted to lymphoid cells, as shown with TCR-␤ and ␤-globin constructs transfected in HeLa cells. Collectively, the data suggest that the ptc-mediated mRNA decay pathway requires an unstable protein, a ribosome, or a ribosome-like entity. Protein synthesis inhibitors may be useful tools toward elucidating the molecular mechanism of ptc-mediated mRNA decay, an enigmatic response that can occur in the nuclear fraction of mammalian cells. T-cell receptor (TCR)1 and immunoglobulin (Ig) genes undergo programmed rearrangement events during lymphocyte ontogeny. During this process, variable (V) elements are juxtaposed to joining (J) elements to create functional genes (1, 2).In some TCR and Ig genes, diversity (D) elements are also included in this rearrangement process. The tremendous combinatorial possibilities afforded by this rearrangement mechanism permit the generation of a wide variety of antigen receptors. Additional variability is provided by the enzyme terminal transferase which introduces random nucleotides at the junctions between V, D, and, J elements (1, 2). Variability is also engendered by the low fidelity of the rearrangement event itself; the borders of each element are not fixed, sometimes leading to small deletions at the junctions between the V, D, and J elements. The collective result of these insertional and deletional events is that a large fraction of rearrangement events will generate out-of-frame (nonproductive) genes that contain premature termination codons (ptcs).Since out-of-frame TCR and Ig genes are commonly generated during normal lymphocyte development, there may exist a mechanism that diminishes the expression of these nonfunctional ptc-containing genes. Consistent with...

Although most eukaryotic mRNAs need a functional cap binding complex eIF4F for efficient 5 end-dependent scanning to initiate translation, picornaviral, hepatitis C viral, and a few cellular RNAs have been shown to be translated by internal ribosome entry, a mechanism that can operate in the presence of low levels of functional eIF4F. To identify cellular mRNAs that can be translated when eIF4F is depleted or in low abundance and that, therefore, may contain internal ribosome entry sites, mRNAs that remained associated with polysomes were isolated from human cells after infection with poliovirus and were identified by using a cDNA microarray. Approximately 200 of the 7000 mRNAs analyzed remained associated with polysomes under these conditions. Among the gene products encoded by these polysome-associated mRNAs were immediate-early transcription factors, kinases, and phosphatases of the mitogen-activated protein kinase pathways and several protooncogenes, including c-myc and Pim-1. In addition, the mRNA encoding Cyr61, a secreted factor that can promote angiogenesis and tumor growth, was selectively mobilized into polysomes when eIF4F concentrations were reduced, although its overall abundance changed only slightly. Subsequent tests confirmed the presence of internal ribosome entry sites in the 5 noncoding regions of both Cyr61 and Pim-1 mRNAs. Overall, this study suggests that diverse mRNAs whose gene products have been implicated in a variety of stress responses, including inflammation, angiogenesis, and the response to serum, can use translational initiation mechanisms that require little or no intact cap binding protein complex eIF4F.

Premature termination codons (PTCs) can cause the decay of mRNAs in the nuclear fraction of mammalian cells. This enigmatic nuclear response is of interest because it suggests that translation signals do not restrict their effect to the cytoplasm, where fully assembled ribosomes reside. Here we examined the molecular mechanism for this putative nuclear response by using the T‐cell receptor‐beta (TCR‐beta) gene, which acquires PTCs as a result of programmed rearrangements that occur during normal thymic ontogeny. We found that PTCs had little or no measurable effect on TCR‐beta pre‐mRNA levels, but they sharply depressed TCR‐beta mature mRNA levels in the nuclear fraction of stably transfected cells. A PTC split by an intron was able to trigger the down‐regulatory response, implying that PTC recognition occurs after an mRNA is at least partially spliced. However, intron deletion and addition studies demonstrated that a PTC must be followed by at least one functional (spliceable) intron to depress mRNA levels. One explanation for this downstream intron‐dependence is that cytoplasmic ribosomes adjacent to nuclear pores scan mRNAs still undergoing splicing as they emerge from the nucleus. We found this explanation to be unlikely because PTCs only 8 or 10 nt upstream of a terminal intron down‐regulated mRNA levels, even though this distance is too short to permit PTC recognition in the cytoplasm prior to the splicing of the downstream intron in the nucleus. Collectively, the results suggest that nonsense codon recognition may occur in the nucleus.