Effects of intron length on differential processing of mouse mu heavy-chain mRNA.

Tsurushita, Naoya; Korn, Laurence Jay

doi:10.1128/mcb.7.7.2602

Cited by 41 publications

(29 citation statements)

References 28 publications

(16 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The size of the intron that is in competition with a poly(A) site has been shown to affect the pA/splice ratio in the C (16,31,32,48), C␣ (41), and D d (unpublished data) genes; when the intron size was decreased, the pA/splice ratio also decreased. Thus, while previous deletions were larger (Ͼ350 nt), it was possible that some of the effect of the NH deletion was due to a change in the intron size.…”

Section: Resultsmentioning

confidence: 99%

An RNA Polymerase Pause Site Is Associated with the Immunoglobulin μs Poly(A) Site

Peterson

Bertolino

Davis

2002

Molecular and Cellular Biology

View full text Add to dashboard Cite

Immunoglobulin alternative RNA processing is regulated during B-cell maturation and requires balanced efficiencies of the competing splice (m) and cleavage-polyadenylation (s) reactions. When we deleted sequences 50 to 200 nucleotides beyond the s poly(A) site, the s/m mRNA ratio decreased three-to eightfold in B, plasma, and nonlymphoid cells. The activity could not be localized to a smaller fragment but did function in heterologous contexts. Our data suggest that this region contains an RNA polymerase II pause site that enhances the use of the s poly(A) site. First, known pause sites replaced the activity of the deleted fragment. Second, the fragment, when placed between tandem poly(A) sites, enhanced the use of the upstream poly(A) site. Finally, nuclear run-ons detected an increase in RNA polymerase loading just downstream from the s poly(A) site, even when the poly(A) site was inactivated. When this fragment and another pause site were inserted 1 kb downstream from the s poly(A) site, they no longer affected the mRNA expression ratio, suggesting that pause sites affect poly(A) site use over a limited distance. Fragments from the immunoglobulin A gene were also found to have RNA polymerase pause site activity.Elements that modulate gene expression by altering RNA polymerase elongation (reviewed in references 9, 35, 43, and 49) have been identified near the 5Ј end of genes (e.g., myc and human immunodeficiency virus) (18,20,26,27), within the coding region of genes (e.g., histone H3.3 and apolipoprotein A-I) (22,37,46), and downstream from some (3, 14), but not all (51), cleavage-polyadenylation [poly(A)] sites. The elements found downstream from the ␣ globin (14) and complement C2 (3) poly(A) sites were shown to contribute to transcriptional termination. The ␣ globin element is contained within a 92-bp fragment that is located about 300 nucleotides (nt) downstream from the ␣ globin poly(A) site (14). The C2 element is a 156-bp fragment that begins just downstream from the C2 poly(A) site; despite its close proximity, it is not required for efficient use of the C2 poly(A) site (3). These elements were called RNA polymerase pause sites because they appeared to have a kinetic affect on RNA polymerase II transcription (3, 14); when placed between two competing reactions, their effect on gene expression was consistent with these elements causing RNA polymerase to pause or slow its elongation rate. This was true when they were placed between tandem poly(A) sites (3,14), between tandem promoters (13), and between a regulated exon and its intronic regulatory region (38). In addition, in nuclear run-on assays, which measure RNA polymerase loading along a transcription unit, an increase in RNA polymerase loading was seen over these elements, again suggesting that RNA polymerase was being slowed or temporarily paused, so there was a higher probability of finding RNA polymerase molecules over those regions (3,14). More recently, the activity of these pause sites and their effect on upstream poly(A) sites was examined in...

show abstract

Section: Resultsmentioning

confidence: 99%

An RNA Polymerase Pause Site Is Associated with the Immunoglobulin μs Poly(A) Site

Peterson

Bertolino

Davis

2002

Molecular and Cellular Biology

View full text Add to dashboard Cite

show abstract

“…These examples include the tissue-specific expression of calcitonin versus CGRP mRNA (14) and the developmental expression of E3 versus L4 mRNA in the adenovirus (1). Work by a number of groups (4,24,30,31) and most recently by Peterson and Perry (25) (26). Our study demonstrates that the 1,243-bp region alone is insufficient to direct the termination process.…”

mentioning

confidence: 81%

Parameters that govern the regulation of immunoglobulin delta heavy-chain gene expression.

Tisch¹,

Kondo²,

Hozumi³

1990

Mol. Cell. Biol.

View full text Add to dashboard Cite

The ,u and 8 immunoglobulin heavy-chain genes comprise a complex transcriptional unit in which a single mRNA precursor gives rise to ,u-and 8-specific transcripts. During the immature B-cell stage, posttranscriptional processing events involving alternate splicing and cleavage-polyadenylation site selection give rise to Jibut not 8-encoding transcripts. In terminally differentiated B cells, 8 mRNA is not synthesized because of a transcription termination event occurring upstream of the 8-gene locus. In an attempt to gain insight into the respective contributions of alternate splicing and cleavage-polyadenylation in the control of 8 mRNA synthesis, we have constructed a set of plasmids in which membrane ,L (l.m)-B intergenic sequences containing the ILm poly(A) site but differing in splicing capacity were inserted in between a VH and 8 gene. The .m4-8 insertion vectors were transfected into a B lymphoma line representative of an immature stage, and proximal ILm poly(A) site usage and 8 mRNA synthesis were assessed. To determine unequivocally whether the p1,-8 intergenic region can regulate termination, the insertion vectors were also transfected into a B myeloma line, and transcription through the region was measured. In immature B-cell transfectants, splicing site selection was found to have a key role in determining poly(A) site utilization and concomitant 8 mRNA expression. Mature 8 mRNA synthesis was blocked by an upstream cleavage-polyadenylation event only when the proximal poly(A) site was associated with appropriate splicing signals. Furthermore, in vitro transcription assays revealed that the iLm-8 intergenic region is sufficient to regulate transcription termination within a 1,243-base-pair region containing the Lm poly(A) site in myeloma transfectants. The Lm-t8 insertion vectors provide an excellent model system for studying the regulatory aspects of this transcription termination event.

show abstract

“…The balance between the two is reflected in the final sec-to-mb mRNA ratio and could potentially be controlled by regulation of either event. However, when the efficiency of splicing of the last constant-region exon to M1 exons was examined in either the or ␥ gene, there was no change in early versus late-stage B cells or in lymphoid versus nonlymphoid cell lines (3,38,40,56), indicating that splicing is a constitutive, nonregulated step. In contrast, experiments using stable transfections have shown that regulated expression of the mouse Ig ␥2b gene is influenced by poly(A) site order and strength (26).…”

mentioning

confidence: 99%

Regulation of Poly(A) Site Use during Mouse B-Cell Development Involves a Change in the Binding of a General Polyadenylation Factor in a B-Cell Stage-Specific Manner

Edwalds-Gilbert

Milcarek

1995

Molecular and Cellular Biology

View full text Add to dashboard Cite

Immunoglobulin (Ig) heavy-chain proteins exist in two forms, differing only at their carboxyl termini. The membranebound antigen receptor is found on the surface of early or memory B cells, while the secreted form is produced by plasma cells. The mRNAs for the two protein forms are derived from a single pre-mRNA that is alternatively processed at the 3Ј end (1,8,44,45). The 3Ј end of the secretion-specific (sec) mRNA is encoded by the last constant-region exon; the sec poly(A) site is about 100 nucleotides downstream of the last constantregion exon. The 3Ј end of the membrane-specific (mb) mRNA is encoded by a large portion of the last constant-region exon and by two downstream exons, M1 and M2. mb mRNA is produced when splicing of the last constant-region exon to M1 takes place using a 5Ј splice site within the last constant-region exon and polyadenylation occurs at the poly(A) site at the end of M2.The ratio of sec to mb mRNA is indicative of a B cell's developmental stage (reviewed in reference 18). Early B, pre-B, and memory cells and their tumor analogs, lymphomas, make approximately equal amounts of sec and mb mRNA. Plasma cells and their tumor counterparts, myelomas, make 10-to 100-fold more sec mRNA than mb mRNA. While translational and posttranslational control mechanisms play a small role in contributing to the sec-versus-mb protein ratios of Igproducing cells, the major contribution to the phenotype comes from differential production of sec and mb mRNAs (13,24,25,41). Studies of the mouse Ig ␥2b locus have shown that transcription termination occurs downstream of the mb poly(A) site in both early and late-stage B cells (11). This finding is in contrast to some studies of the Ig transcription unit (13,19), in which differential transcription termination has been suggested as a possible mechanism for the switch in poly(A) site use during B-cell development. The stability of both ␥ sec and ␥ mb mRNAs increases in late-stage or plasma cells, and differential mRNA stability therefore cannot account for the major shift in the ratio of the two mRNAs (33).The key point in understanding posttranscriptional processing of Ig heavy-chain mRNAs is that splicing of the last constant-region exon to M1 and polyadenylation at the sec poly(A) site are two mutually exclusive events. The balance between the two is reflected in the final sec-to-mb mRNA ratio and could potentially be controlled by regulation of either event. However, when the efficiency of splicing of the last constant-region exon to M1 exons was examined in either the or ␥ gene, there was no change in early versus late-stage B cells or in lymphoid versus nonlymphoid cell lines (3,38,40,56), indicating that splicing is a constitutive, nonregulated step. In contrast, experiments using stable transfections have shown that regulated expression of the mouse Ig ␥2b gene is influenced by poly(A) site order and strength (26).The biochemistry of cleavage and polyadenylation and the sequences required in cis in the pre-mRNA for 3Ј-end formation have been well chara...

show abstract

Effects of intron length on differential processing of mouse mu heavy-chain mRNA.

Cited by 41 publications

References 28 publications

An RNA Polymerase Pause Site Is Associated with the Immunoglobulin μs Poly(A) Site

An RNA Polymerase Pause Site Is Associated with the Immunoglobulin μs Poly(A) Site

Parameters that govern the regulation of immunoglobulin delta heavy-chain gene expression.

Regulation of Poly(A) Site Use during Mouse B-Cell Development Involves a Change in the Binding of a General Polyadenylation Factor in a B-Cell Stage-Specific Manner

Contact Info

Product

Resources

About