Subcellular localization of RNAs in transfected cells: role of sequences at the 5′ terminus

Alfonso-Pizarro, Aixa; Carlson, David P.; Ross, Jeffrey

doi:10.1093/nar/12.22.8363

Cited by 10 publications

(9 citation statements)

References 75 publications

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“…The 5' end located 131 bp upstream from the BglII site probably represents initiation from the tk promoter, although tk mRNA is initiated at a different site during an HSV infection. The use of upstream transcription initiation sites during transient expression of genes introduced into Cos-1 cells has been reported previously by others (1,42).…”

Section: Resultsmentioning

confidence: 68%

Identification of sequences in the herpes simplex virus thymidine kinase gene required for efficient processing and polyadenylation.

Cole¹,

Stacy²

1985

Mol. Cell. Biol.

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The herpes simplex virus (HSV) type 1 thymidine kinase gene (tk) was resected from its 3' end with BAL 31 exonuclease. Two sets of plasmids were isolated that lacked information distal to the two copies of the hexanucleotide 5'-AATAAA-3' located at the 3' end of the HSV tk gene. The presence of a simian virus 40 origin of DNA replication in each plasmid facilitated analysis of patterns of transcription in transfected Cos-1 monkey cells. Transcription analyses were performed with an Si nuclease protection assay. Efficient processing and polyadenylation at the normal site still occurred when all sequences more than 44 or 46 base pairs (bp) downstream from the first AATAAA were removed (pTK311R/SVO10 and pTK209R/SVO10). Removal of an additional 7 bp (pTK312R/SVO10) decreased the amount of tk mRNA processed at that normal site, and tk mRNA polyadenylated at a cryptic site within pBR322 sequences began to appear. The normal processing and polyadenylation site was not used at all when an additional 12 bp was removed (pTK314R/SVO10); the small amount of tk mRNA produced was processed and polyadenylated at the cryptic pBR322 site. The region of the tk gene critical for efficient processing and polyadenylation of tk mRNA is located 20 to 38 bp downstream from the first AATAAA, distal to the polyadenylation site, and as RNA can form a stem-loop structure containing AAUAAA. Similar G+T-rich elements were located in DNA fragments which substitute efficiently for the HSV tk processing and polyadenylation signal and were not found in AATAAA-containing DNA fragments which substitute inefficiently for the HSV tk signal.The biosynthesis of eucaryotic mRNA requires a series of processing steps which convert the precursor heterogeneous nuclear RNA into functional mRNA (see reference 38 for a review). These processing steps include the addition of caps at the 5' end of the mature mRNA, the removal of intervening sequences by splicing, and the addition of polyadenylic acid at the 3' end of the mature mRNA. Although transcription by RNA polymerase II initiates at the cap site, evidence from several systems (17, 18,22,39) indicates that transcription extends beyond the site of polyadenylation and that endonucleolytic cleavage creates the site at which a poly(A) polymerase adds approximately 200 A residues.

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Section: Resultsmentioning

confidence: 68%

Identification of sequences in the herpes simplex virus thymidine kinase gene required for efficient processing and polyadenylation.

Cole¹,

Stacy²

1985

Mol. Cell. Biol.

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“…lanes 3 and 4). Since release of mRNPs from polysomes in the presence of EDTA is consistent with their active translation (2,40), these results suggest that cytoplasmic variant IV mRNA is exported to the cytoplasm, associates with polysomes, and may be translated. While variant V mRNA was also detected in the cytoplasmic 130,000 ϫ g pellet, its retention in the presence of EDTA raises the possibility that it is contained within a translationally inactive complex (28) or that it may localize to an EDTA-insensitive structure such as the cytoskeleton (20,22) or membranous components (29).…”

Section: Vol 19 1999 Alternative Splicing In the Auf1 3ј-utr 4059mentioning

confidence: 56%

Regulation of AUF1 Expression via Conserved Alternatively Spliced Elements in the 3′ Untranslated Region

Wilson

Sun

Sellers

et al. 1999

Molecular and Cellular Biology

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The A؉U-rich RNA-binding factor AUF1 exhibits characteristics of a trans-acting factor contributing to the rapid turnover of many cellular mRNAs. Structural mapping of the AUF1 gene and its transcribed mRNA has revealed alternative splicing events within the 3 untranslated region (3-UTR). In K562 erythroleukemia cells, we have identified four alternatively spliced AUF1 3-UTR variants, including a population of AUF1 mRNA containing a highly conserved 107-nucleotide (nt) 3-UTR exon (exon 9) and the adjacent downstream intron (intron 9). Functional analyses using luciferase-AUF1 3-UTR chimeric transcripts demonstrated that the presence of either a spliceable or an unspliceable intron 9 in the 3-UTR repressed luciferase expression in cis, indicating that intron 9 sequences may down-regulate gene expression by two distinct mechanisms. In the case of the unspliceable intron, repression of luciferase expression likely involved two AUF1-binding sequences, since luciferase expression was increased by deletion of these sites. However, inclusion of the spliceable intron in the luciferase 3-UTR down-regulated expression independent of the AUF1-binding sequences. This is likely due to nonsense-mediated mRNA decay (NMD) owing to the generation of exon-exon junctions more than 50 nt downstream of the luciferase termination codon. AUF1 mRNA splice variants generated by selective excision of intron 9 are thus also likely to be subject to NMD since intron 9 is always positioned >137 nt downstream of the stop codon. The distribution of alternatively spliced AUF1 transcripts in K562 cells is consistent with this model of regulated AUF1 expression. The cytoplasmic steady-state level of any given mRNA, and hence its potential for translation, is a cumulative function of its rates of synthesis (transcription), nuclear pre-mRNA processing, nucleocytoplasmic transport, and cytoplasmic decay. Thus, the potential exists for cis-acting elements to regulate the abundance of individual mRNAs at many stages (reviewed in reference 6). Genetic determinants regulating the rate of mRNA turnover are frequently localized to the 3Ј untranslated region (3Ј-UTR) (reviewed in references 4, 36, and 38).AϩU-rich elements (AREs) are cis-acting determinants of rapid cytoplasmic mRNA turnover found in the 3Ј-UTRs of many constitutively labile transcripts, including some encoding oncoproteins, inflammatory mediators, cytokines, and G-protein-coupled receptors (reviewed in references 10 and 36). AREs are variable in length but frequently consist of a number of overlapping AUUUA pentamers located within or adjacent to a U-rich region. While sequence and functional heterogeneity is observed among these destabilizing elements from different mRNAs, ARE-directed mRNA turnover is generally characterized by rapid deadenylation followed by decay of the mRNA body. Since many ARE-containing mRNAs encode essential components of pathways regulating cell growth, inflammation, and immune responses, the control of mRNA decay through these sequences influences many biologica...

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“…(48), dihydrofolate reductase (23), ferritin (11), the globin gene family (2,16), and rat brain mRNAs (44); however, the roles of such alternative or multiple cap sites and the resulting untranslated 5' mRNA sequences are unknown. It has been proposed that differences in the 5' cap site and untranslated sequences regulate transcriptional stability and translational efficiency (1,18). That the proteoglycan mRNA differences in 5' untranslated sequences may have a functional significance is suggested by the development and cell-type-specific expression of the two PG19 mRNA forms, a pattern also observed for the alcohol dehydrogenase gene of D. melanogaster (4) and the mouse a-amylase gene (48).…”

Section: Discussionmentioning

confidence: 97%

Gene expression of the chondroitin sulfate proteoglycan core protein PG19.

Bourdon¹,

Shiga²,

Ruoslahti³

1987

Mol. Cell. Biol.

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We have examined genomic sequences and mRNA species hybridizing to a cDNA clone of a yolk sac carcinoma chondroitin sulfate proteoglycan designated PG19. Genomic blot hybridizations with cDNAs covering the majority of the PG19 mRNA sequence revealed 15 to 17 gene fragments. Similar analysis with probes representing either the propeptide or the combined core protein COOH-terminal domain and 3' untranslated sequences revealed single genomic fragments indicating that a single gene codes for the PGl9 proteoglycan. Genomic blot analysis with cDNA sequences coding for the serine-glycine repeat of the core protein identified the same gene fragments observed with the entire PG19 cDNA, indicating that this coding region is homologous with sequences present in multiple genes. The same probes were also used to examine mRNA expression. In addition to the PG19 mRNA, several PG19-related mRNAs could be seen. These PG19-related mRNAs had homology with the serine-glycine coding sequence of the PG19 cDNA. These mRNAs may be coding for proteoglycans. The mRNA coding for PG19 appeared to be uniquely expressed in parietal yolk sac and mast cell lineages. The PG19 mRNA existed in different forms in parietal yolk sac and mast cell lines due to cell-type-specific differences in the length of the 5' untranslated sequences. These results indicate that expression of the PG19 proteoglycan gene is regulated both in terms of cell-type-specific transcription and selection of a transcriptional start site.Proteoglycans are protein glycoconjugates present in a variety of basement membranes (17,20,30), extracellular matrices (3,8,17,26,31), and cell membranes (21, 34). Functionally and structurally, little is known about proteoglycans beyond their glycoconjugate structure (17). It is likely, however, that the diversity of core proteins revealed by biochemical and immunological studies and, more recently, sequence analyses of cloned cDNAs (6, 6a, 12, 21a, 35) reflects functional diversity as well. As examples, one proteoglycan appears to represent a subpopulation of the transferrin receptor (14), and another is associated with the intracellular transport of class II histocompatibility antigen polypeptides (37). Other proteoglycans interact with the major extracellular matrix and basement membrane components such as fibronectin (29), vitronectin (42), collagen (21, 29), and laminin (36, 47) and in doing so may regulate the formation of extracellular matrices and the adhesion of cells to such matrices (10, 21). The modulation of cell adhesion in particular may be important for cell division and cell migration, especially in malignant cells (10, 34).We have been interested in the structure and expression of a chondroitin sulfate proteoglycan secreted by a yolk sac carcinoma cell line (27) because of its interaction with extracellular matrix proteins (7, 29) and its ability to inhibit yolk sac carcinoma cell adhesion to collagen and fibronectin (7). The amino acid sequence of the yolk sac carcinoma proteoglycan core protein precursor and its proces...

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Subcellular localization of RNAs in transfected cells: role of sequences at the 5′ terminus

Cited by 10 publications

References 75 publications

Identification of sequences in the herpes simplex virus thymidine kinase gene required for efficient processing and polyadenylation.

Identification of sequences in the herpes simplex virus thymidine kinase gene required for efficient processing and polyadenylation.

Regulation of AUF1 Expression via Conserved Alternatively Spliced Elements in the 3′ Untranslated Region

Gene expression of the chondroitin sulfate proteoglycan core protein PG19.

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