RNA processing is a limiting step for murine tumor necrosis factor beta expression in response to interleukin-2.

Abstract

We have previously reported that tumor necrosis factor I8 (TNFO) expression is induced by interleukin-2 (IL-2) in the murine lymphocytic T-cell line CTLL-2. In this study, we have characterized the nuclear and cytoplasmic TNFi transcripts and assessed their role in TNF(8 gene expression. A unique feature of TNFji expression was the accumulation of nuclear precursors, which reflected a slow nuclear RNA processing. As a consequence, there was a delay in the appearance of cytoplasmic messengers after the transcriptional induction of TNF,3 by IL-2. We also found that two messengers, the fully spliced messenger and an intron 3-retaining messenger, were exported to the cytoplasm and actively translated. The same pattern of expression was observed in concanavalin A-stimulated splenocytes, although the level of expression was much lower than in CTLL-2 cells. The simple genetic structure and the high level of accumulation of nuclear precursors make TNF,3 a particularly attractive model system to use for studies of RNA processing and cytoplasmic transport of partially spliced messengers.

Mature mRNAs accumulated in the nucleus are neither the molecules in transit to the cytoplasm nor constitute a stockpile for gene expression

“…Rabbit b-globin and murine LT-a were chosen to investigate the behavior of the nuclear pool of mature mRNAs after arrest of transcription+ Both genes are not naturally expressed in fibroblasts, and their expression in transfected NIH3T3 cells has been previously characterized (Fig+ 1; Neel et al+, 1995)+ b-globin contains two introns, both of them being spliced out efficiently; the cytoplasmic half-life of b-globin mRNA has been reported to be on the order of 11 h in fibroblastic cells (Schiavi et al+, 1994)+ LT-a contains three introns, which are sequentially removed according to a 59-to-39 polarity (Weil et al+, 1990)+ In addition, splicing of intron 3 is slower than that of introns 1 and 2+ Fully spliced mRNA (designated as N0 in Fig+ 1) as well as intron 3-containing transcripts (designated as N1 in Fig+ 1) are exported to the cytoplasm+ Both cytoplasmic transcripts (C0 and C1 in Fig+ 1) have a half-life of 90 min in the T lymphocytic cell line CTLL-2 (Weil et al+, 1990)+ For both genes the complete transcribed region, including the introns, was inserted in an expression vector under the control of a Tet promoter (Dirks et al+, 1994)+ These vectors were stably transfected into the NIHtTA-16-3 cell line (a kind gift of A+ Kröger) that constitutively expresses the chimeric VP16-tTA transactivator allowing for negative regulation by tetracycline (Tet-off system) (Gossen & Bujard, 1992)+ Independent clones were isolated and screened for their level of expression of b-globin or LT-a mRNA+ Clones with a high expression level in the absence of tetracycline (hereafter designated tTA-b-glo and tTA-LT-a) were further characterized+…”

“…Regulation of LT-a expression by tetracycline+ A: RNase protection assay analysis+ tTA-LT-a cells were treated with indicated tetracycline concentrations for 20 h+ Equal amounts of nuclear (N) and cytoplasmic (C) RNA were analyzed using the LT-a probe represented in Figure 1+ Migration of the riboprobe fragments protected by partially and fully spliced transcripts is indicated on the right+ See Figure 1 for nomenclature of the transcripts+ Unexpected protected fragments are indicated with asterisks+ B: Quantification of cytoplasmic LT-a mRNA+ LT-a signals were quantified using a Molecular Dynamics Storm analyzer, corrected for the number of labeled uridine in each fragment, and plotted as a function of tetracycline concentration+ Concentration of mRNA samples was first checked by Northern blot analysis with b-actin probe+ Note that both axes are on a logarithmic scale+ experiment was performed on tTA-LT-a cells (Fig+ 3A, Tet 0 mg/mL)+ Fully spliced LT-a mRNA was more abundant in the nuclear fraction than in the cytoplasmic one (1+8-fold), so that 17% of the fully mature mRNA of the cell is nuclear+ In contrast to b-globin, and as previously described (Weil et al+, 1990;Neel et al+, 1995), LT-a partially spliced transcripts (N2, N3, and N1) could be readily observed in the nuclear fraction, indicating limiting rates of splicing+ For comparison, the nucleocytoplasmic partition of an endogenous gene was also analyzed and 17% of actin mRNA was found in the nucleus+…”

“…To measure the extent of regulation of promoter activity by tetracycline, tTA-b-glo cells were cultured in the presence of different doses of tetracycline for 72 h so that a new steady state had been reached+ This incubation time corresponds to ten cytoplasmic half-lives for b-globin mRNA (see below)+ Cytoplasmic and nuclear RNA was extracted and analyzed by Northern blot (Fig+ 2A)+ Quantification of cytoplasmic b-globin mRNA indicated that repression of b-globin expression was maximal with 1 mg/mL tetracycline, leading to a 120-fold decrease in mRNA accumulation (Fig+ 2B)+ Intermediate levels of expression could be achieved using 2-60 ng/mL tetracycline+ The same experiment was performed on tTA-LT-a cells using an incubation of 20 h in the presence of tetracycline, a time corresponding to ten cytoplasmic half-lives for LT-a mRNA+ As the short size of LT-a intron 3 (225 nt long) does not allow for proper resolution of the two cytoplasmic transcripts (C0 and C1) on Northern blot, LT-a transcripts were analyzed by an RNase protection assay using a probe extending from exon 2 to exon 4 (Weil et al+, 1990) (Fig+ 3A)+ Maximal repression was achieved with 1 mg/mL tetracycline and resulted in an 18-fold decrease in fully spliced mRNA (C0 in Fig+ 3B) and a 7-fold decrease in intron 3-containing transcripts (C1 in Fig+ 3B)+ Unexpected fragments, indicated by asterisks, were also detected+ They correspond to abnormal transcripts and, because their expression followed that of normal transcripts, their identity was not investigated further+ The 120-fold repression of b-globin and the 18-fold repression of LT-a expression provided the opportunity to investigate the dynamics of the nuclear pool of mature mRNAs after inhibition of transcription by tetracycline+…”

Regulation of mRNA splicing and transport by the tyrosine kinase activity of src

“…This gene constitutes a convenient reporter for premRNA processing studies (Neel et al, 1993(Neel et al, , 1995Weil et al, 1990) because of its short size, moderate level of splicing complexity (three introns which are sequentially removed from the polyadenylated primary transcript), ine cient splicing of intron 3 and the export to the cytoplasm of intron 3 containing precursors as well as fully mature mRNA ( Figure 1b). RNase protection assays can be used to unambiguously identify the di erent RNA species and reliably quantitate their accumulation.…”

Genes encoding the β1 subunit of voltage‐dependent Na+ channel in rat, mouse and human contain conserved introns

“…Several cases of intron retention has been reported in the literature [28][29][30][31]. Retained introns could maintain the open reading frame and result in an alternate protein [29,30].…”