Position and sequence requirements for poly(A) length regulation by the poly(A) limiting element

Gupta, Jaydip Das; Gu, Howard H.; Schoenberg, Daniel R.

doi:10.1017/s1355838201010329

Cited by 7 publications

(12 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Transcription run-on was done next to determine if this increase resulted from enhanced reporter gene transcription. In the experiment in Figure 1C nuclei isolated from cells transfected with each of these genes or a b-globin reporter gene bearing an inactivated PLE (MutG) (Das Gupta et al 2001) were incubated with a-[ 32 P]UTP, and serial twofold dilutions of the resulting radiolabeled transcripts were hybridized to filters containing an excess of filter-bound b-globin or luciferase cDNAs. Hybridized radioactivity was quantified by PhosphorImager analysis and results with each dilution set were compared.…”

Section: Resultsmentioning

confidence: 99%

“…Upstream elements often modulate the efficiency of pre-mRNA 3 0 processing (Zhao et al 1999), and the fact that the PLE must be in the last exon to regulate poly(A) tail length (Das Gupta et al 2001) raised the possibility that it might also enhance the efficiency of 3 0 processing. The first experiment to test this used an RT-PCR approach similar to that used by Juge et al (2002) to examine the role of poly(A) polymerase on cleavage efficiency in vivo.…”

Section: Resultsmentioning

confidence: 99%

“…The location of the PLE close to AAUAAA in the constructs used for the preceding experiments complicated efforts to design a single probe that would hybridize to each reporter mRNA. To address this we took advantage of previous work showing that additional sequence can be inserted between the PLE and AAUAAA without affecting PLE regulation of poly(A) length (Das Gupta et al 2001). …”

Section: Resultsmentioning

confidence: 99%

“…PLE B and MutG elements were inserted as doublestranded oligonucleotides into the multiple cloning site between the stop codon and the SPA. A PLE-containing plasmid with an additional 200 bp of fX174 DNA (Das Gupta et al 2001) was digested with XbaI to generate a 158-bp fragment that was inserted into the XbaI site of each of the b-globin reporter genes to generate the plasmids used in Figure 4. To prepare the luciferase control (CMVluc-SPA) CMV-glo-SPA was digested with Asp718 and end filled with Klenow fragment of DNA polymerase followed by digestion with NcoI to remove the globin cassette, leaving intact the CMV promoter and SPA 3 0 processing element.…”

Section: Plasmid Constructsmentioning

confidence: 99%

“…Deletion analysis identified two redundant PLEs in the terminal exon of albumin pre-mRNA (Das Gupta et al 1998), either of which changed the length of the poly(A) tail on a human b-globin reporter mRNA from $200 nt to <20 nt when placed upstream of a highly efficient synthetic polyadenylation element (SPA) (Levitt et al 1989;Das Gupta et al 1998). To function in regulating poly(A) length the PLE must be in the terminal exon, and as long this location is maintained the distance between it and AAUAAA could be changed without affecting its ability to restrict poly(A) to <20 nt (Das Gupta et al 2001).…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

The poly(A)-limiting element enhances mRNA accumulation by increasing the efficiency of pre-mRNA 3′ processing

Peng

Murray²,

Schoenberg³

2005

RNA

Self Cite

View full text Add to dashboard Cite

The poly(A)-limiting element (PLE) is a conserved sequence originally found in the 3 0 UTR of Xenopus albumin mRNA whose presence restricts the length of the poly(A) tail on both pre-mRNA and fully processed mRNA to <20 nt. Results presented in this study show that the PLE also increases the cytoplasmic level of reporter b-globin mRNA. Transcription run-on shows this increase was not due to increased reporter gene transcription, and experiments with tetracycline repressor-controlled reporter mRNA showed the PLE does not alter the rate of mRNA decay. Both RT-PCR and RNase protection assay showed the PLE caused a 50% increase in the 3 0 processing of reporter b-globin mRNA in vivo. This was confirmed in vitro, where PLE-containing RNA was cleaved in HeLa nuclear extract at a rate 80% faster than a control RNA bearing an inactive element. These results indicate that the PLE regulates the length of the poly(A) tail and the efficiency of 3 0 processing. In addition, they show that PLE-containing mRNA with a <20-nt poly(A) tail is as stable as mRNA with a 100-to 200-nt poly(A) tail.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Plasmid Constructsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

The poly(A)-limiting element enhances mRNA accumulation by increasing the efficiency of pre-mRNA 3′ processing

Peng

Murray²,

Schoenberg³

2005

RNA

Self Cite

View full text Add to dashboard Cite

show abstract

Regulation of alternative RNA splicing by exon definition and exon sequences in viral and mammalian gene expression

2004

View full text Add to dashboard Cite

Intron removal from a pre-mRNA by RNA splicing was once thought to be controlled mainly by intron splicing signals. However, viral and other eukaryotic RNA exon sequences have recently been found to regulate RNA splicing, polyadenylation, export, and nonsense-mediated RNA decay in addition to their coding function. Regulation of alternative RNA splicing by exon sequences is largely attributable to the presence of two major cis-acting elements in the regulated exons, the exonic splicing enhancer (ESE) and the suppressor or silencer (ESS). Two types of ESEs have been verified from more than 50 genes or exons: purine-rich ESEs, which are the more common, and non-purine-rich ESEs. In contrast, the sequences of ESSs identified in approximately 20 genes or exons are highly diverse and show little similarity to each other. Through interactions with cellular splicing factors, an ESE or ESS determines whether or not a regulated splice site, usually an upstream 3' splice site, will be used for RNA splicing. However, how these elements function precisely in selecting a regulated splice site is only partially understood. The balance between positive and negative regulation of splice site selection likely depends on the cis-element's identity and changes in cellular splicing factors under physiological or pathological conditions.

show abstract

Purifying mRNAs with a high-affinity eIF4E mutant identifies the short 3′ poly(A) end phenotype

Choi

Hagedorn

2003

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

View full text Add to dashboard Cite

The use of DNA microarrays has revolutionized the manner in which mRNA populations are analyzed. One limitation of the current technology is that mRNAs are often purified on the basis of their 3 poly(A) ends, which can be extremely short or absent in some mRNAs. To circumvent this limitation, we have developed a procedure for the purification of eukaryotic mRNAs using a mutant version of the mRNA 5 cap-binding protein (eIF4E) with increased affinity for the m 7 GTP moiety of the cap. By using this procedure, we have compared the populations of mammalian mRNAs purified by oligo(dT) and 5 cap selection with oligonucleotide microarrays. This analysis has identified a subpopulation of mRNAs that are present with short 3 poly(A) ends at steady state and are missed or underrepresented after purification by oligo(dT). These mRNAs may respond to specific posttranscriptional control mechanisms such as cytoplasmic polyadenylation. Initiation of eukaryotic mRNA translation requires the recruitment of many proteins, the initiator tRNA, and a 40S ribosomal subunit to the 5Ј cap of mRNA. Eukaryotic initiation factor 4E (eIF4E or 4E) specifically recognizes the m 7 GDP moiety of the m 7 G(5Ј)ppp(5Ј)N cap present at the 5Ј end of eukaryotic mRNAs (1-3). In addition to binding the 5Ј cap of eukaryotic mRNAs, 4E also binds the ribosome adaptor protein (4G) and translational repressor proteins (4EBPs or PHAS I/II) and associates indirectly with poly(A)-binding protein to stimulate translation (4-9). 4E is a common point of growth regulation in both untransformed and cancer cells (10-13). Both structural and functional studies provide evidence that 4E binds the m 7 G moiety of the 5Ј cap of mRNAs by a -stacking interaction between two tryptophan residues, as well as hydrogen bonds between m 7 G and acidic side chains of 4E (14-17). We have recently described mutants in the S4-H2 loop of 4E (N118A, K119A, and Q120A) that had an affinity for m 7 GTP several-fold increased relative to wild-type 4E (18).DNA microarray technology has enabled new questions to be identified or answered in a wide variety of biological systems (19). Although total RNA can be analyzed by DNA microarrays to quantitate gene expression, purification of mRNA with oligo(dT) can frequently decrease problems with signal-to-noise ratios, and the analysis of specific subsets of mRNAs can provide useful information (20). Wild-type 4E fused to protein A has been used previously to isolate eukaryotic mRNAs by binding their 5Ј caps (21). We tested the use of a high-affinity mutant of 4E to purify mRNA and determine how it compared with oligo(dT) and whether it might more effectively isolate a subset of eukaryotic mRNAs. One of these 4E mutants (4E K119A ) enabled the high-yield 5Ј cap-dependent purification of functional mRNAs from total RNA. Moreover, an unexpectedly large number of mRNAs were more efficiently isolated by using high-affinity 4E compared with oligo(dT), suggesting that they had short 3Ј poly(A) ends that diminished their binding to oligo(dT). This mRNA p...

show abstract

Position and sequence requirements for poly(A) length regulation by the poly(A) limiting element

Cited by 7 publications

References 29 publications

The poly(A)-limiting element enhances mRNA accumulation by increasing the efficiency of pre-mRNA 3′ processing

The poly(A)-limiting element enhances mRNA accumulation by increasing the efficiency of pre-mRNA 3′ processing

Regulation of alternative RNA splicing by exon definition and exon sequences in viral and mammalian gene expression

Purifying mRNAs with a high-affinity eIF4E mutant identifies the short 3′ poly(A) end phenotype

Contact Info

Product

Resources

About