Identification of a stem-loop structure important for polyadenylation at the murine IgM secretory poly(A) site

Phillips, Cathy; Kyriakopoulou, Christina; Virtanen, Anders

doi:10.1093/nar/27.2.429

Cited by 25 publications

(35 citation statements)

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“…Although, within these two groups, the secondary structures formed by the NUE and the CS signals vary, the range of the variation is reasonably within the predicted signal regions. The stem loop structure has been observed in relation to many poly(A) events, where the CSs are situated on the loop, and mutation of such by base substitution or deletion severed cleavage activities, leading to decreased biological activity as characterized in the IgM secretory transcript (Phillips et al, 1999). This phenomenon may be explained by loss of recognition and formation of the CPSF/CstF complex due to mutation or signal loss (Phillips et al, 1999).…”

Section: Discussionmentioning

confidence: 99%

“…The stem loop structure has been observed in relation to many poly(A) events, where the CSs are situated on the loop, and mutation of such by base substitution or deletion severed cleavage activities, leading to decreased biological activity as characterized in the IgM secretory transcript (Phillips et al, 1999). This phenomenon may be explained by loss of recognition and formation of the CPSF/CstF complex due to mutation or signal loss (Phillips et al, 1999). Many examples involving hairpins, internal loops, and globular circles represent target sites for RNA-interacting protein (Ruff et al, 1991).…”

Section: Discussionmentioning

confidence: 99%

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Compilation of mRNA Polyadenylation Signals in Arabidopsis Revealed a New Signal Element and Potential Secondary Structures

et al. 2005

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Using a novel program, SignalSleuth, and a database containing authenticated polyadenylation [poly(A)] sites, we analyzed the composition of mRNA poly(A) signals in Arabidopsis (Arabidopsis thaliana), and reevaluated previously described cis-elements within the 3#-untranslated (UTR) regions, including near upstream elements and far upstream elements. As predicted, there are absences of high-consensus signal patterns. The AAUAAA signal topped the near upstream elements patterns and was found within the predicted location to only approximately 10% of 3#-UTRs. More importantly, we identified a new set, named cleavage elements, of poly(A) signals flanking both sides of the cleavage site. These cis-elements were not previously revealed by conventional mutagenesis and are contemplated as a cluster of signals for cleavage site recognition. Moreover, a singlenucleotide profile scan on the 3#-UTR regions unveiled a distinct arrangement of alternate stretches of U and A nucleotides, which led to a prediction of the formation of secondary structures. Using an RNA secondary structure prediction program, mFold, we identified three main types of secondary structures on the sequences analyzed. Surprisingly, these observed secondary structures were all interrupted in previously constructed mutations in these regions. These results will enable us to revise the current model of plant poly(A) signals and to develop tools to predict 3#-ends for gene annotation.Messenger RNA polyadenylation is a crucial step during the maturation of most eukaryotic mRNA, in which a polyadenine [poly(A)] tract is added to the cleaved 3#-end of a precursor mRNA (pre-mRNA) posttranscriptionally. Such a modification of mRNA has been shown to affect its stability, translatability, and nuclear-to-cytoplasmic export (Zhao et al., 1999). The posttranscriptional processing of mRNA is an event that has also been found tightly coupled with splicing and transcription termination (Proudfoot et al., 2002;Proudfoot, 2004). Thus, it is an essential processing event and the integral part of gene expression.The polyadenylation process requires two major components: the cis-elements or poly(A) signals of the pre-mRNA, and the trans-acting factors that carry out the cleavage and addition of the poly(A) tail at the 3#-end. These trans-acting factors are a complex of about 25 to 30 proteins involved in signal recognition, cleavage, and polyadenylation (Proudfoot, 2004). These proteins seem to be conserved among eukaryotic organisms. However, the poly(A) signals have been found to differ widely among yeast (Saccharomyces cerevisiae), animals, and plants in terms of signal locations and sequence content. The highly conserved AAUAAA element in mammals becomes a minor signal in plant and yeast genes, and the ubiquitous downstream elements of mammalian pre-mRNAs are nowhere to be found in yeast and plants. The latter two possess an enhancing element located far upstream of the cleavage site (Zhao et al., 1999).Previous understanding of these signal elements was derived mostly...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Compilation of mRNA Polyadenylation Signals in Arabidopsis Revealed a New Signal Element and Potential Secondary Structures

et al. 2005

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“…According to principles of antisense technology the kinetics and thermodynamics of oligonucleotide hybridization are profoundly attenuated when hybridization has to overcome energy barriers caused by secondary RNA structure (37,39). Hence lower hybridization re¯ects more stable secondary RNA structures (32). Thus the ribosome binding sites of the wildtype mRNAs were stabilized by the introduction of the RNA stem±loop.…”

Section: Structural Changes Of Regulatory Elements On the Mrna By Intmentioning

confidence: 99%

“…Single-stranded RNA is accessible to hybridization and allows cleavage, while double-stranded RNA stretches are not accessible and therefore remain uncleaved by RNase H (30±32). Additionally, hybridization of increasing amounts of oligonucleotides competes against native RNA structure at the targeted site and demonstrates the accessibility, and hence stability, of the secondary RNA structure (32,37).…”

Section: Formation Of the Introduced Rna Stem±loopmentioning

confidence: 99%

RNA stem-loop enhanced expression of previously non-expressible genes

Paulus¹

2004

Nucleic Acids Research

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The key step in bacterial translation is formation of the pre-initiation complex. This requires initial contacts between mRNA, fMet-tRNA and the 30S subunit of the ribosome, steps that limit the initiation of translation. Here we report a method for improving translational initiation, which allows expression of several previously non-expressible genes. This method has potential applications in heterologous protein synthesis and high-throughput expression systems. We introduced a synthetic RNA stem±loop (stem length, 7 bp; DG 0 = ±9.9 kcal/mol)

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“…The presence of negative regulatory elements has also been described. In certain contexts, inhibition of polyadenylation was found to be associated with the presence of a splice donor site located either upstream or downstream of the poly(A) site (11,18,25,26). For example, insertion of a bona fide 5Ј-splice site between the 3Ј-splice site and the poly(A) site was shown to abolish the coupling of the two reactions both in vivo and in vitro (27).…”

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confidence: 99%

The Cleavage/Polyadenylation Activity Triggered by a U-rich Motif Sequence Is Differently Required Depending on the Poly(A) Site Location at Either the First or Last 3′-Terminal Exon of the 2′-5′ Oligo(A) Synthetase Gene

Aissouni¹,

Pérez²,

Calmels³

et al. 2002

Journal of Biological Chemistry

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Production of the two mRNAs encoding distinct forms of 2-5-oligoadenylate synthetase depends on processing that involves the recognition of alternative poly(A) sites and an internal 5-splice site located within the first 3-terminal exon. The resulting 1.6-and 1.8-kb mRNAs are expressed in fibroblast cell lines, whereas lymphoblastoid B cells, such as Daudi, produce only the 1.8-kb mRNA. In the present study, we have shown that the 3-end processing at the last 3-terminal exon occurs independently of the core poly(A) site sequence or the presence of regulatory elements. In contrast, in Daudi cells, the recognition of the poly(A) site at the first 3-terminal exon is impaired because of an unfavorable sequence context. The 3-end processing at this particular location requires a strong stabilization of the cleavage/polyadenylation factors, which can be achieved by the insertion of a 25-nucleotide long U-rich motif identified upstream of the last poly(A) site. Consequently, we speculate that in cells expressing the 1.6-kb mRNA, such as fibroblasts, direct or indirect participation of a specific mechanism or cell type-specific factors are required for an efficient polyadenylation at the first 3-terminal exon.Polyadenylation of nearly all eukaryotic pre-mRNAs is an obligatory step in the maturation of transcripts (reviewed in Ref. 1). The 3Ј-end processing occurs by cleavage of the precursor RNA, generating the upstream cleavage product that is elongated by a poly(A) tail (for review, see Refs. 2 and 3). The core elements of a polyadenylation signal correspond to a highly conserved hexanucleotide AAUAAA found 10 -30 nucleotides upstream of the cleavage site and a less highly conserved GU-rich element located downstream of the cleavage site. Cleavage/polyadenylation is an intricate process requiring multiple cellular factors. At the first step of the polyadenylation reaction, the GU-rich sequence is recognized by one component of the cleavage stimulating factor (CstF), 1 the protein CstF-64, which stabilizes the binding of the cleavage/polyadenylation specificity factor (CPSF) to the AAUAAA hexamer (2). Other factors that are required are the cleavage factors (CFI m , CFII m ) (4, 5) and poly(A) polymerase. Finally, the Poly(A) binding protein II (PABII) specifies the correct length of the poly(A) tail and increases the efficiency of polyadenylation (6 -8), enhancing mRNA stability and translation (9, 10).Besides the core elements and secondary structures that influence cleavage/polyadenylation (11), auxiliary sequences located either upstream or downstream of poly(A) sites have been reported to influence 3Ј-end processing efficiency in a positive or negative manner. Upstream sequence enhancers (USEs) were primarily identified in viral poly(A) sites (12-16) and further in poly(A) sites of cellular genes, such as those encoding the complement factor C2 (17), lamin B2 (18, 19), and the secretory form of IgM (20). For these genes, the presence of USEs that are often U-rich increases polyadenylation efficiency. In contrast t...

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Identification of a stem-loop structure important for polyadenylation at the murine IgM secretory poly(A) site

Cited by 25 publications

References 50 publications

Compilation of mRNA Polyadenylation Signals in Arabidopsis Revealed a New Signal Element and Potential Secondary Structures

Compilation of mRNA Polyadenylation Signals in Arabidopsis Revealed a New Signal Element and Potential Secondary Structures

RNA stem-loop enhanced expression of previously non-expressible genes

The Cleavage/Polyadenylation Activity Triggered by a U-rich Motif Sequence Is Differently Required Depending on the Poly(A) Site Location at Either the First or Last 3′-Terminal Exon of the 2′-5′ Oligo(A) Synthetase Gene

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