Sequences Adjacent to the 5′ Splice Site Control U1A Binding Upstream of the IgM Heavy Chain Secretory Poly(A) Site

Phillips, Chris; Gunderson, Samuel

doi:10.1074/jbc.m301349200

Cited by 12 publications

(11 citation statements)

References 36 publications

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“…Indeed, U1A has been shown to inhibit both 3Ј cleavage and polyadenylation of the IgM heavy chain mRNA during B cell differentiation, and, similarly to the SMN system, U1A binds to the IgM heavy chain pre-mRNA both upstream and downstream of the regulated cleavage site. U1A may be functioning on the SMN mRNA in a manner similar to the IgM heavy chain system (47,66,67). Identifying the molecular details of the inhibitory mechanism presents an interesting avenue for further investigation.…”

Section: Discussionmentioning

confidence: 99%

U1A Regulates 3′ Processing of the Survival Motor Neuron mRNA

Workman

Veith

Battle

2014

Journal of Biological Chemistry

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Background: Insufficient expression of survival motor neuron (SMN), a protein involved in small nuclear ribonucleoprotein (snRNP) biogenesis, causes spinal muscular atrophy (SMA). Results: The U1A protein binds to and inhibits polyadenylation and cleavage of the SMN pre-mRNA. Conclusion: The U1A protein is a critical regulator of SMN expression. Significance: Understanding of the regulation of SMN expression is fundamental for developing treatments for SMA.

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Section: Discussionmentioning

confidence: 99%

U1A Regulates 3′ Processing of the Survival Motor Neuron mRNA

Workman

Veith

Battle

2014

Journal of Biological Chemistry

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“…A possible protection downstream of the cleavage site had previously been noticed while investigating U1A's ability to protect the upstream U1A binding sites in the context of an RNA substrate spanning 295 nucleotides (nt), position 1790 to 2085 encompassing a possible cloverleaf structure (19). To investigate the downstream footprints more closely, we 3Ј end-labeled the substrate (position 1790 to 2085) (for location sequence and motifs spanned, see protection from partial digestion with RNase T1 (which cuts 3Ј of accessible guanosines) (7).…”

Section: Resultsmentioning

confidence: 99%

“…However, these bands were not very intense, suggesting low-affinity binding without the surrounding sequences. As long-range RNA-RNA interactions control the accessibility of the upstream sites (19), RNA outside the 1924 to 2030 region may contribute to the structure of these sites and their accessibility to U1A.…”

Section: Resultsmentioning

confidence: 99%

U1A Inhibits Cleavage at the Immunoglobulin M Heavy-Chain Secretory Poly(A) Site by Binding between the Two Downstream GU-Rich Regions

Phillips

Pachikara

Gunderson

2004

Molecular and Cellular Biology

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The immunoglobulin M heavy-chain locus contains two poly(A) sites which are alternatively expressed during B-cell differentiation. Despite its promoter proximal location, the secretory poly(A) site is not expressed in undifferentiated cells. Crucial to the activation of the secretory poly(A) site during B-cell differentiation are changes in the binding of cleavage stimulatory factor 64K to GU-rich elements downstream of the poly(A) site. What regulates this change is not understood. The secretory poly(A) site contains two downstream GU-rich regions separated by a 29-nucleotide sequence. Both GU-rich regions are necessary for binding of the specific cleavage-polyadenylation complex. We demonstrate here that U1A binds two (AUGCN 1-3 C) motifs within the 29-nucleotide sequence and inhibits the binding of cleavage stimulatory factor 64K and cleavage at the secretory poly(A) site.The immunoglobulin M (IgM) heavy-chain pre-mRNA () is alternatively processed into mRNAs encoding a membrane receptor or secreted antibody during differentiation (4) (Fig. 1). It is the classic model for an important pattern of alternative processing which involves competition between splicing and cleavage-polyadenylation, and it includes a number of important receptors involved in growth and differentiation (for a review, see reference 6). In undifferentiated cells, exons encoding a membrane tail are spliced on and the mRNA is cleaved at a downstream, membrane poly(A) site, resulting in mRNA encoding the heavy chain of the membrane receptor. When cells differentiate into Ig-secreting cells, an upstream, secretory poly(A) site is activated within the intron involved in the splicing of the membrane exons. This results in the secretory form of mRNA which encodes the heavy chain of a secreted antibody. The secretory form of -mRNA is expressed in differentiated cells by a combination of increased cleavage at the secretory poly(A) site and increased stability of the secretory mRNA itself (1,3,10,12).3Ј end cleavage in metazoans takes place on the recognition of a bipartite poly(A) signal consisting of a consensus AAUAAA and a less-defined GU-rich sequence, upstream and downstream of the cleavage site, respectively, by components of the cleavage-polyadenylation complex. These consist of the multimeric cleavage polyadenylation specificity factor (CPSF) and cleavage stimulatory factor (CstF), of which the 64-kDa component (CstF64K) recognizes the GU-rich region, as well as cleavage factors I and II and poly(A) polymerase (reviewed in reference 31). After cleavage, the RNA is specifically polyadenylated by poly(A) polymerase tethered to the RNA via the 160-kDa component of CPSF, bound by the AAUAAA sequence (16).

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“…Non-U1snRNP U1A functions in a multiprotein complex to regulate polyadenylation, further suggesting a link between splicing and polyadenylation. U1A has also been shown to be important for the switch from membrane-bound to secreted Immunoglobulin M (IgM) that occurs during B-cell differentiation (Phillips et al 2001(Phillips et al , 2004Phillips and Gunderson 2003;Ma et al 2006). In immature B-cells, free U1A inhibits the polyadenylation of secretory IgM by binding to elements that resemble degenerate consensus U1A binding sequences (A(U/G)GC(N 1-3 )C) in the IgM pre-mRNA (Phillips et al 2001(Phillips et al , 2004Phillips and Gunderson 2003;Ma et al 2006).…”

Section: Implications Of Single Stranded Rna Binding By U1amentioning

confidence: 99%

The role of RNA structure in the interaction of U1A protein with U1 hairpin II RNA

Law

Rice

Lin

et al. 2006

RNA

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The N-terminal RNA Recognition Motif (RRM1) of the spliceosomal protein U1A interacting with its target U1 hairpin II (U1hpII) has been used as a paradigm for RRM-containing proteins interacting with their RNA targets. U1A binds to U1hpII via direct interactions with a 7-nucleotide (nt) consensus binding sequence at the 59 end of a 10-nt loop, and via hydrogen bonds with the closing C-G base pair at the top of the RNA stem. Using surface plasmon resonance (Biacore), we have examined the role of structural features of U1hpII in binding to U1A RRM1. Mutational analysis of the closing base pair suggests it plays a minor role in binding and mainly prevents ''breathing'' of the loop. Lengthening the stem and nontarget part of the loop suggests that the increased negative charge of the RNA might slightly aid association. However, this is offset by an increase in dissociation, which may be caused by attraction of the RRM to nontarget parts of the RNA. Studies of a single stranded target and RNAs with untethered loops indicate that structure is not very relevant for association but is important for complex stability. In particular, breaking the link between the stem and the 59 side of the loop greatly increases complex dissociation, presumably by hindering simultaneous contacts between the RRM and stem and loop nucleotides. While binding of U1A to a single stranded target is much weaker than to U1hpII, it occurs with nanomolar affinity, supporting recent evidence that binding of unstructured RNA by U1A has physiological significance.

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Sequences Adjacent to the 5′ Splice Site Control U1A Binding Upstream of the IgM Heavy Chain Secretory Poly(A) Site

Cited by 12 publications

References 36 publications

U1A Regulates 3′ Processing of the Survival Motor Neuron mRNA

U1A Regulates 3′ Processing of the Survival Motor Neuron mRNA

U1A Inhibits Cleavage at the Immunoglobulin M Heavy-Chain Secretory Poly(A) Site by Binding between the Two Downstream GU-Rich Regions

The role of RNA structure in the interaction of U1A protein with U1 hairpin II RNA

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