Sequence discrimination by alternatively spliced isoforms of a DNA binding zinc finger domain

Wild-type Chlamydomonas reinhardtii cells shifted from high concentrations (5%) of CO 2 to low, ambient levels (0.03%) rapidly increase transcription of mRNAs from several CO 2-responsive genes. Simultaneously, they develop a functional carbon concentrating mechanism that allows the cells to greatly increase internal levels of CO 2 and HCO 3 ؊ . The cia5 mutant is defective in all of these phenotypes. A newly isolated gene, designated Cia5, restores transformed cia5 cells to the phenotype of wild-type cells. The 6,481-bp gene produces a 5.1-kb mRNA that is present constitutively in light in high and low CO 2 both in wild-type cells and the cia5 mutant. It encodes a protein that has features of a putative transcription factor and that, likewise, is present constitutively in low and high CO 2 conditions. Complementation of cia5 can be achieved with a truncated Cia5 gene that is missing the coding information for 54 C-terminal amino acids. Unlike wild-type cells or cia5 mutants transformed with an intact Cia5 gene, cia5 mutants complemented with the truncated gene exhibit constitutive synthesis of mRNAs from CO 2-responsive genes in light under both high and low CO 2 conditions. These discoveries suggest that posttranslational changes to the C-terminal domain control the ability of CIA5 to act as an inducer and directly or indirectly control transcription of CO 2-responsive genes. Thus, CIA5 appears to be a master regulator of the carbon concentrating mechanism and is intimately involved in the signal transduction mechanism that senses and allows immediate responses to fluctuations in environmental CO 2 and HCO 3 ؊ concentrations.

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“…Two motifs within the CIA5 protein have clear similarities to zinc finger motifs found in numerous transcription factors (21,22) (Fig. 1C).…”

Section: Discussionmentioning

confidence: 99%

The Cia5 gene controls formation of the carbon concentrating mechanism in Chlamydomonas reinhardtii

Xiang

Zhang

Weeks

2001

Proc. Natl. Acad. Sci. U.S.A.

135

117

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“…However, some mRNAs encoding transcription factors are subjected to regulated splicing. This can produce mRNAs encoding different transcription factors with distinct functions (51)(52)(53)(54)(55). Natural and artificially incomplete transcription factors (lacking the activation domain) are able to interact with complete factors and act as negative dominants (44 -47, 56).…”

Section: Discussionmentioning

confidence: 99%

The Product of the Rice myb7 Unspliced mRNA Dimerizes with the Maize Leucine Zipper Opaque2 and Stimulates Its Activity in a Transient Expression Assay

Locatelli

Bracale²,

Magaraggia³

et al. 2000

Journal of Biological Chemistry

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myb7 mRNA is present in rice in spliced and unspliced forms, splicing being enhanced by anoxia. The protein (Mybleu) encoded by the unspliced mRNA is composed of an incomplete Myb domain followed by a leucine zipper; however, it lacks canonical sequences for DNA binding, transcriptional activation, and nuclear localization. We show here that in transiently transformed tobacco protoplasts, Mybleu is able to enhance the transcriptional activity of the maize leucine zipper Opaque2 on its target b32 promoter. The Mybleu transactivation effect is strictly dependent on the presence of Opaque2 and is driven by Mybleu-Opaque2 heterodimers. Mybleu is located in the nucleus, both in rice and in transformed tobacco protoplasts. In rice, the protein is expressed in regions corresponding to undifferentiated cells of roots and coleoptiles. Therefore, myb7 mRNA encodes, depending on its splicing, two transcription factors belonging to separate classes. One of them, Mybleu, has novel structural characteristics, suggesting the existence of new mechanisms acting in the activation of transcription.Mybs are a family of transcription factors widely represented in viruses, insects, mammals, and plants. The common feature of Myb factors is the presence of a conserved domain consisting of imperfect repeats of 50 -53 amino acids, called "tryptophan clusters" because of the highly conserved tryptophan residues involved in stabilizing the structure of the DNA binding domain (1). In plants, myb genes are present as large families (6 -100 members) involved in the control of a wide range of biochemical pathways (2-6), including responses to biotic and abiotic stresses (7-13).Screening a cDNA library from anaerobically grown rice coleoptiles, we isolated myb7, a cDNA derived from a Mybencoding unspliced mRNA (8). Some features of myb7 sequence indicate that it may be post-transcriptionally regulated; in particular, two unspliced introns are present in positions that are conserved with respect to other plant myb genes. Both spliced and unspliced myb7 mRNAs are present in vivo (7).In aerobically grown rice roots, we observed a higher proportion of unspliced myb7 RNA, with respect to the spliced form. The spliced form is instead predominant in rice roots during anoxia, a stress situation that generally inhibits splicing (14). These data suggest that post-transcriptional regulation controls the ratio between myb7 unspliced and spliced forms (7). This hypothesis is supported by the observation that the 5Ј region of the first intron can direct the synthesis of an in-frame leucine zipper, a functional domain present in several transcription factors (15-18).The putative polypeptide encoded by the unspliced myb7 mRNA consists of an incomplete Myb domain followed by the leucine zipper: we therefore named it Mybleu. Mybleu does not have the characteristics of a functional transcription factor. Indeed, it should be unable to bind DNA because of the presence of an incomplete Myb repeat. Moreover, neither a putative transcriptional activator region nor a c...

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“…PCR products and Gfi-1 protein were incubated in EMSA binding buffer [4 mM Tris-HCl (pH 7.5), 80 mM NaCl, 0.5 mM ZnSO 4 , 1 mM EDTA, 0.5 mM DTT, 1 g of poly(dI-dC), 1 g of poly(dA-dT) (Sigma), 5% glycerol] at 22ЊC for 30 min. Samples were electrophoresed for ϳ3 h at 100 V on prerun, nondenaturing polyacrylamide gels (6% [29:1] acryl-bisacrylamide, 0.25ϫ TBE, 5% glycerol) (18). To determine the relative importance of individual zinc fingers for binding to the Gfi-1 consensus, we generated a series of zinc finger deletion mutants in which all of the zinc fingers were deleted one at a time (⌬ZnF1 to ⌬ZnF6).…”

Section: Methodsmentioning

confidence: 99%

Gfi-1 Encodes a Nuclear Zinc Finger Protein That Binds DNA and Functions as a Transcriptional Repressor

Zweidler‐McKay

Grimes

Flubacher

et al. 1996

Molecular and Cellular Biology

278

285

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The Gfi-1 proto-oncogene encodes a zinc finger protein with six C 2 H 2 -type, C-terminal zinc finger motifs and is activated by provirus integration in T-cell lymphoma lines selected for interleukin-2 independence in culture and in primary retrovirus-induced thymomas. Gfi-1 expression in adult animals is restricted to the thymus, spleen, and testis and is enhanced in mitogen-stimulated splenocytes. In this report, we show that Gfi-1 is a 55-kDa nuclear protein that binds DNA in a sequence-specific manner. The Gfi-1 binding site, TAAATCAC(A/ T)GCA, was defined via random oligonucleotide selection utilizing a bacterially expressed glutathione S-transferase-Gfi-1 fusion protein. Binding to this site was confirmed by electrophoretic mobility shift assays and DNase I footprinting. Methylation interference analysis and electrophoretic mobility shift assays with mutant oligonucleotides defined the relative importance of specific bases at the consensus binding site. Deletion of individual zinc fingers demonstrated that only zinc fingers 3, 4, and 5 are required for sequence-specific DNA binding. Potential Gfi-1 binding sites were detected in a large number of eukaryotic promoter-enhancers, including the enhancers of several proto-oncogenes and cytokine genes and the enhancer of the human cytomegalovirus (HCMV) major immediate-early promoter, which contains two such sites. HCMV major immediate-early-chloramphenicol acetyltransferase reporter constructs, transfected into NIH 3T3 fibroblasts, were repressed by Gfi-1, and the repression was abrogated by mutation of critical residues in the two Gfi-1 binding sites. These results suggest that Gfi-1 may play a role in HCMV biology and may contribute to oncogenesis and T-cell activation by repressing the expression of genes that inhibit these processes.

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Sequence discrimination by alternatively spliced isoforms of a DNA binding zinc finger domain

Cited by 97 publications

References 22 publications

The Cia5 gene controls formation of the carbon concentrating mechanism in Chlamydomonas reinhardtii

The Cia5 gene controls formation of the carbon concentrating mechanism in Chlamydomonas reinhardtii

The Product of the Rice myb7 Unspliced mRNA Dimerizes with the Maize Leucine Zipper Opaque2 and Stimulates Its Activity in a Transient Expression Assay

Gfi-1 Encodes a Nuclear Zinc Finger Protein That Binds DNA and Functions as a Transcriptional Repressor

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