Alternative production of calcitonin and CGRP mRNA is regulated at the calcitonin-specific splice acceptor

Yee

Journal of Biological Chemistry

2004

The Rous sarcoma virus (RSV) negative regulator of splicing (NRS) is an RNA element that represses splicing and promotes polyadenylation of viral RNA. The NRS acts as a pseudo 5 splice site (ss), and serine-arginine (SR) proteins, U1snRNP, and U6 small nuclear ribonucleoproteins (snRNPs) are implicated in its function. The NRS also efficiently binds U11 snRNP of the U12-dependent splicing pathway, which is interesting, because U11 binds only poorly to authentic substrates that lack a U12-type 3 splice site. It is of considerable interest to understand how the low abundance U11 snRNP binds the NRS so well. Here we show that U11 can bind the NRS as a mono-snRNP in vitro and that a G-rich element located downstream of the U11 site is required for efficient binding. Mutational analyses indicated that two of four G tracts in this region were important for optimal U11 binding and that the G-rich region did not function indirectly by promoting U1 snRNP binding to an overlapping site. Surprisingly, inactivation of U2 snRNP also decreased U11 binding to the NRS. The NRS harbors a branch point-like/pyrimidine tract sequence (BP/Py) just upstream of the U1/U11 site that is characteristic of 3 splice sites. Deletion of this region decreased U2 and U11 binding, and deletion of the G-rich region also reduced U2 binding. The G element, but not the BP/Py sequence, was also required for U11 binding to the NRS in vivo as assessed by minor class splicing from the NRS to a minor class 3ss from the P120 gene. These results indicate that efficient U11 binding to the isolated NRS involves at least two elements in addition to the U11 consensus sequence and may have implications for U11 binding to authentic splicing substrates.An important step in the production of most mRNAs is the removal of intron sequences from pre-mRNA by the process of RNA splicing. Different combinations of exons can be combined via alternative splicing to generate numerous proteins from a single gene (1), and it is now appreciated that regulated splicing accounts for great protein diversity (1-3). RNA splicing is also used by many viruses that infect eukaryotic cells as a means to expand their coding capacity and to regulate gene expression (4). This is true for retroviruses, with HIV 1 being an extreme example: it is estimated that more than 40 viral splice variants are produced in an HIV-infected cell (5-7). In addition to alternative splicing, retroviruses also exhibit incomplete splicing such that a large fraction of the primary transcripts remain unspliced and, in contrast to unspliced host cell mRNAs, are transported to the cytoplasm where they serve as mRNA and as genomes for progeny virions. These RNA processing events make retroviruses useful tools for studying the cellular RNA processing machinery, and determining how these events are controlled is required for understanding these important aspects of viral replication. RNA splicing takes place in a large macromolecular complex termed the spliceosome in which five small nuclear ribonucleoprotein partic...

Section: Discussionmentioning

confidence: 82%

Two Regions Promote U11 Small Nuclear Ribonucleoprotein Particle Binding to a Retroviral Splicing Inhibitor Element (Negative Regulator of Splicing)

Yee

Journal of Biological Chemistry

2004

“…These cell lines and others were also assayed for src splicing by RNase protection with similar results (data not shown), indicating that the ratio of the two product bands has not been skewed by the PCR amplification process and also that the upper bands in the HeLa lane are not derived from src m-RNA. Another well-studied neural-specific splicing event, in the calcitonin/CGRP gene, occurs efficiently in the embryonal carcinoma cell line F9 (Emeson et al 1989). As these cells make little or no n -s r c (data not shown), these two neural-specific splicing patterns are likely to be regulated by different factors.…”

Section: Cell Lines That Express N-srcmentioning

confidence: 99%

Does steric interference between splice sites block the splicing of a short c-src neuron-specific exon in non-neuronal cells?

Black¹

1991

Genes Dev.

139

132

The neuron-specific splicing of the mouse c-src N1 exon was analyzed. Model src genes, transiently expressed in HeLa and LA-N-5 neuroblastoma cells, were assayed for the insertion of the 18-nucleotide neuron-specific N1 exon into their product mRNA. The normal clone fails to use this exon in HeLa cells but inserts the exon into 50% of the mature mRNA in LA-N-5 cells. When the exon and flanking intron sequences are placed between two adenovirus exons, the N1 exon is still only inserted in the neural cells. Thus, the neural specificity is a property of the exon itself and its immediate flanking sequences. Simply extending the length of the N1 exon to 109 nucleotides allows its efficient use in HeLa cells, implying that the exon is normally skipped because it is too short to allow spliceosomes to assemble at both ends simultaneously. This model predicts that exclusion of the exon should be sensitive to proteins or mutations that alter the relative strength of the flanking splice sites. Mutations that change these splice sites support this hypothesis.[Key Words: Pre-mRNA splicing~ alternative splicing~ neurons~ c-src; neural src~ spliceosomes]

Journal of Biological Chemistry

“…RNase Protection Assays and in Situ Hybridization Analysis-RNase protection assays were performed using 32 P-radiolabeled probes, as described previously (19). Hybridization reactions contained 10 g of yeast tRNA or total tissue RNA.…”

Section: Methodsmentioning

confidence: 99%

A Novel Family of Cys-Cys, His-Cys Zinc Finger Transcription Factors Expressed in Developing Nervous System and Pituitary Gland

Jiang

Buchholz

et al. 1996

Self Cite

113

A screen designed to identify proteins that specifically bind to retinoic acid response elements resulted in the identification of a rat cDNA encoding a novel protein containing six Cys-Cys, His-Cys zinc fingers. This gene is expressed in a restricted fashion exhibiting distinct temporal and spatial patterns in the developing nervous system, primarily brain, spinal cord, sensory ganglia, retina, and nasal epithelia, as well as in the pituitary, and is referred to as neural zinc finger factor 1 (NZF-1). NZF-1 binds specifically to a cis-regulatory element of the ␤-retinoic acid receptor (RAR␤) gene, as well as to other related DNA elements, including two in the upstream enhancer region of the mouse Pit-1 gene. In heterologous cells, NZF-1 activates transcription from promoters containing specific binding sequences and can synergize with other factors, such as Pit-1, to regulate gene expression. These results suggest that NZF-1 may exert regulatory roles in the developing and mature nervous system and in the pituitary gland. Identification of a second mouse gene highly homologous to NZF-1, encoded by a distinct genomic locus, reveals a dispersed gene family encoding proteins containing Cys-Cys, His-Cys motifs.Precise temporal and spatial patterns of development are controlled by sequential activation of a hierarchy of regulatory genes, which encode transcription factors containing multiple classes of DNA binding motifs. Zinc coordinated fingers are one of the most common DNA binding motifs among eukaryotic transcription factors and are classified based on amino acid sequence of the zinc fingers. The Cys-Cys, His-His class, which is typified by the Xenopus transcription factor IIIA (1), contains the largest number of members. These proteins contain two or more fingers in a tandem repeat. In contrast, steroid receptors, such as the glucocorticoid receptor, contain only two zinc coordinated structures with four (C 4 ) and five (C 5 ) conserved cysteines. The third class of zinc fingers, which also binds to single-stranded nucleic acids, has a consensus sequence of Cys-X 2 -Cys-X 4 -His-X 4 -Cys. Such factors are found in transposable element copia, plants, and mammalian cells as well as in retroviruses. Other metal-coordinating proteins have different structures such as C 6 in the yeast GAL4 protein and a cysteinerich structure in the E1A oncoprotein (2).In accordance with their structural diversity, zinc finger proteins play a variety of important roles in cell growth, differentiation, and development. Transcription factor IIIA and the ubiquitous transcription factor SP1 are broadly involved in the regulation of transcription, whereas the Drosophila zinc finger proteins Krü ppel and Hunchback are crucial for proper segmentation of the developing embryo (3-5). In humans, mutations in a kidney zinc finger protein (WT1) result in Wilm's tumor (6, 7). Recently, a zinc finger protein (REST) has been shown to repress neuronal gene expression in non-neuronal tissues (8, 9).Because retinoic acid receptor (RAR) 1 binds ineff...