Splicing Mechanisms That Generate Distinct Isoforms of Protein 4.1R During Terminal Erythroid Differentiation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4036-4036
Author(s):  
Marilyn K Parra ◽  
Narla Mohandas ◽  
John G. Conboy
Keyword(s):  
Splice Site ◽  
Conflicts Of Interest ◽  
Protein Isoforms ◽  
Pcr Analysis ◽  
Acceptor Site ◽  
Intermediate Structure ◽  
Splice Acceptor ◽  
Exon 2 ◽  
Regulatory Motifs ◽  
Protein 4.1R

Abstract Abstract 4036 Poster Board III-972 The protein 4.1R gene is a large complex gene with two translation initiation sites (AUG1 and AUG2) that encode protein isoforms with distinct N-terminal structure and function. Expression of these isoforms is regulated by alternative splicing at either of two splice acceptor sites that flank exon 2' (E2'), in which AUG1 is located. In late erythroblasts E2' is excluded, ensuring translation at AUG2 and synthesis of 80kDa protein 4.1R isoforms. Our earlier studies (EMBO J. 27:122-31, 2008) described a two-step intrasplicing pathway that enforces this splicing outcome exclusively in 4.1R pre-mRNA initiated at exon 1A (E1A), the major transcription start site in late erythropoiesis. The downstream exon 1B (E1B) first splices to the proximal acceptor site at E2' to generate an intermediate structure, which is then re-processed by splicing of E1A to the internal acceptor at E2, removing E1B as well as E2'/AUG1. Experiments with minigenes suggested that intrasplicing is likely independent of specific promoter elements at E1A, but absolutely requires 5' splice site and branch point motifs associated with E1B. Here we sought evidence for functionality of these latter elements in the more physiological context of the endogenous 4.1R gene. The intrasplicing model predicts that morpholino oligonucleotides complementary to key regulatory motifs will block the first step of the pathway in natural 4.1R pre-mRNA transcripts, and yield inappropriate splicing of E1A to the first acceptor at E2'. Antisense morpholinos directed against the E1B branchpoint or E1B 5'splice site were transfected into cells and 4.1R splicing was examined 48hrs later by RT-PCR analysis. Both anti-4.1 morpholinos, but not a control morpholino, resulted in a concentration-dependent shift of E1A splicing to the proximal E2' acceptor site. In other studies we explored whether intrasplicing could occur internally within a gene, using a model pre-mRNA in which a constitutive exon was engineered between E1A and E1B. Analysis of this experiment suggested that any exon upstream of active E1B would follow the intrasplicing pathway and delete E2'. We speculate that internal E1B-like elements in other genes could be selectively activated or silenced by splicing regulatory motifs in order to control downstream splice acceptor choice. Preliminary experiments indicate that weakening the E1B 5' splice site and its upstream pyrimidine tract might permit such regulation as observed for other alternative exons. Together these results provide additional support for the intrasplicing model and suggest that it could function more widely in human genes to coordinate splicing events and generate multiple protein isoforms with distinct functions. Disclosures: No relevant conflicts of interest to declare.

Mechanisms That Link Promoter Choice with Downstream Alternative Splicing in the Erythroid Protein 4.1R Gene.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1562-1562
Author(s):  
Marilyn Parra ◽  
Jeff Tan ◽  
Narla Mohandas ◽  
John G. Conboy
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Rna Processing ◽  
Regulatory Element ◽  
Donor Site ◽  
Splice Acceptor Site ◽  
Acceptor Site ◽  
Splice Acceptor ◽  
Exon 2 ◽  
Protein 4.1R

Abstract The protein 4.1R gene is a large transcription unit (240kb) that utilizes complex RNA processing pathways to encode distinct protein isoforms, both during erythropoiesis and also in nonerythroid cells. Proper regulation of these pathways is essential for stage-specific synthesis of the 80-kDa isoforms of 4.1R protein during terminal erythroid differentiation. The 5′ region of the gene contains multiple alternative first exons that map far upstream of the coding exons, and we have shown previously that promoter choice is coupled to alternative splicing decisions 100kb downstream in exon 2′/2. Transcripts that initiate at exon 1A predominate in late stages of erythropoiesis and splice only to a weak internal 3′ splice acceptor site in exon 2, skipping translation start site AUG1 and ensuring proper translation initiation at AUG2 in exon 4 for synthesis of the 80-kDa isoforms. In contrast, 4.1 transcripts initiated at exons 1B or 1C exclusively splice to the strong first 3′ splice acceptor site at exon 2′ to include AUG1 and encode a higher molecular weight 135-kDa isoform known to interact with different affinity to major erythroid membrane proteins in earlier stages of erythropoiesis. Our studies show that this linkage between transcription and splicing is (a) cell type independent; (b) conserved in the 4.1R gene from fish to man; and (c) conserved in the paralogous 4.1B gene. Our recent functional studies suggest that a novel re-splicing mechanism, reminiscent of recursive splicing of large introns previously described in the Drosophila ubx gene, may couple promoter choice with downstream splicing in the 4.1R gene. Using minigenes that reproduce the differential splicing patterns in transfected mammalian cells, we have shown that accurate splicing of exon 1A requires a unique downstream regulatory element. This element maps several kilobases downstream of exon 1A and is conserved among mammals. Analysis of wild type and mutated minigenes suggests a two step splicing model in which this element behaves as a temporary “intra-exon” that is present in a splicing intermediate but eliminated from the mature mRNA. According to this model, the regulatory element behaves as an exon in the first step as its consensus 5′ donor site splices to the strong 3′ splice site of exon 2′, removing this splice site pair and joining the intra-exon directly to exon 2′. In the second step, the juxtaposed region of the intra-exon then behaves as an intron, contributing to the activation of the weak internal splice acceptor at exon 2. This second splicing event joins exon 1A to exon 2, thus deleting the intra-exon, the 2′ region (and AUG1) and generating a mature 5′ end capable of encoding 80-kDa 4.1R. Importantly, pre-mRNA constructs that lack the intra-exon, or have a mutated intra-exon 5′ splice donor site, are uncoupled and exhibit inappropriate splicing of exon 1A to the first acceptor site at exon 2′. In support of the generality of this model, we have identified a candidate intra-exon with similar sequence properties in the long 5′ region of the human 4.1B gene, and have demonstrated that this element successfully rescues proper splicing of 4.1R exon 1A in our minigenes. Detailed molecular analysis is under way to identify the specific cis and trans elements required to effect this unusual, long-distance coupling between RNA processing events which have implications for detailed mechanistic understanding of membrane assembly during erythropoiesis.

Evolutionarily Conserved Coupling of Transcription and Alternative Splicing in the Protein 4.1R and 4.1B Genes Regulates N-Terminal Protein Structure.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1664-1664
Author(s):  
Jeff Tan ◽  
Marilyn K. Parra ◽  
Narla Mohandas ◽  
John G. Conboy
Keyword(s):  
Alternative Splicing ◽  
Rna Processing ◽  
Regulatory Elements ◽  
Protein Isoforms ◽  
Tissue Type ◽  
Translation Initiation Site ◽  
Acceptor Site ◽  
Exon 2 ◽  
Evolutionarily Conserved ◽  
Protein 4.1R

Abstract The protein 4.1R gene is regulated by complex pre-mRNA processing events that facilitate the synthesis of protein isoforms with different structure, function, and subcellular localization in red cells and various nucleated cell types. One of these events involves the stage-specific activation of exon 16 inclusion in erythroblasts, which mechanically stabilizes the membrane skeleton by increasing the protein’s affinity for spectrin and actin. Some of the splicing factor proteins and RNA regulatory elements responsible for this tissue-specific alternative splicing event have been defined. Here we focus on another RNA processing event, in the 5′ end of the transcript that can affect the structure and function of the membrane binding domain of protein 4.1R. We have shown that 4.1R transcripts originating at three far upstream alternative promoters/first exons splice differentially to alternative acceptor sites in exon 2′/2 in a manner that suggests strict coupling between transcription and alternative splicing events. A precisely analogous gene organization and RNA processing pattern has also been shown to occur in the paralogous 4.1B gene. Now we demonstrate that this coupling is evolutionarily conserved among several vertebrate classes from fish to mammals. The 4.1R and 4.1B genes from fish, bird, amphibian, and mammal genomes exhibit shared features including alternative first exons and differential splice acceptors in exon 2. In all cases, the 5′-most exon (exon 1A) splices exclusively to a weaker internal acceptor site in exon 2, skipping a short sequence designated as exon 2′ (17-33nt). Conversely, alternative first exons 1B and/or 1C always splice to the stronger first acceptor site, retaining exon 2′. These correlations are independent of tissue type or species of origin. Since exon 2′ contains a translation initiation site, this regulated splicing event generate protein isoforms with distinct N-termini. We propose that these 4.1 genes represent a physiologically relevant model system for mechanistic analysis of transcription-coupled alternative splicing. We have recently constructed a 9kb “minigene” that successfully reproduces the differential splicing patterns of exons 1A and 1B to exon 2′/2 in transfected cells. This minigene will facilitate identification of the determinants that guide coupling. Current experiments are testing the importance for proper splicing of the transcriptional promoter, first exon sequences, length and sequence of the intron, and sequence of a conserved element within exon 2′. Ultimately these studies should provide new insights into the mechanisms of coupling between far upstream, transcription-related processes and downstream alternative splicing.

In Vivo Analysis of Erythroid Protein 4.1 Pre-mRNA Splicing Mechanisms: Use of Antisense Morpholinos to Assay Function of Deep Intron Regulatory Elements

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 815-815
Author(s):  
Marilyn Parra ◽  
Xiuli An ◽  
Narla Mohandas ◽  
John G. Conboy
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Cultured Cells ◽  
Conflicts Of Interest ◽  
Regulatory Elements ◽  
Protein Isoforms ◽  
Start Codon ◽  
Exon 2 ◽  
Mouse Tissues

Abstract Abstract 815 Erythroid stage-specific alternative splicing plays an essential role in the expression of protein 4.1R isoforms that interact with other skeletal proteins to strengthen the membrane. In late erythroblasts, 4.1R mRNA is processed from pre-mRNA that initiates transcription at alternative first exon 1A (E1A) and splices exclusively to the more distal of two alternative 3' splice sites at exon 2 (E2dis), ~100kb downstream. This splicing event is important because it is required to generate the shorter N-terminal domain characteristic of 80kDa isoforms of 4.1R protein in red cells. We have reported that E1A splicing to E2dis requires two nested intrasplicing events mediated by an essential deep intron element originally annotated as exon 1B. However, these studies employed small minigenes transfected into cultured cells, an artificial system that may not correctly reflect in vivo mechanisms. Here we used an antisense RNA strategy to explore splicing of endogenous full length 4.1R pre-mRNA in tissues of live mice and in primary erythroblasts. Chemically modified oligonucleotides known as vivo-morpholinos (vMOs), introduced via tail vein injection and internalized into selected organs, can base pair with complementary cellular RNA sequences and block function of candidate regulatory motifs. Importantly, two independent vMOs directed against the 4.1R intraexon regulatory element both substantially abrogated intrasplicing in several mouse tissues, robustly switching E1A splicing from E2dis to the proximal 3' splice site in E2 (E2prox). This switch results in inclusion of start codon AUG1 in mature 4.1R mRNA and synthesis of larger isoforms of 4.1R protein. These results were highly sequence-specific, since negative control vMOs directed against other genes did not alter E1A splicing to E2dis. Interestingly, we have recently used vMOs to confirm the existence of a similar deep intron element required for analogous E1A-E2dis splicing in the paralogous 4.1B gene. Together these findings strongly support the in vivo physiological function of deep intron elements in the control of intrasplicing in both 4.1R and 4.1B pre-mRNAs. To test whether the 4.1R intrasplicing mechanism is also active in erythroid cells, we incubated mouse splenic erythroblasts isolated from FVA-treated animals with morpholinos directed against the intraexon. Two independent morpholinos against its 5' splice site and branch point both induced a concentration-dependent switch in E1A splicing from E2dis to E2prox. Control morpholinos had no effect on E1A splicing. Because the splicing switch results in inclusion of alternative translation initiation codon AUG1, it was predicted to induce synthesis of larger isoforms of 4.1R including the N-terminal headpiece known to influence 4.1R binding affinities for other skeletal proteins. Western blot analysis of erythroblast proteins confirmed a switch to expression of larger 4.1R protein isoforms that are not present in normal late stage erythroblasts. Intrasplicing is mediated by deep intron elements, and is essential for accurate physiological splicing of natural 4.1R pre-mRNA in erythroid and other cells. Antisense morpholinos represent a new tool for alternative splicing studies in vivo or in cultured erythroblasts. Disclosures: No relevant conflicts of interest to declare.

Alternative 5′ exons and differential splicing regulate expression of protein 4.1R isoforms with distinct N-termini

Blood ◽  
2003 ◽  
Vol 101 (10) ◽  
pp. 4164-4171 ◽  
Author(s):  
Marilyn K. Parra ◽  
Sherry L. Gee ◽  
Mark J. Koury ◽  
Narla Mohandas ◽  
John G. Conboy
Keyword(s):  
Alternative Splicing ◽  
Translation Initiation ◽  
Transcription Initiation ◽  
Genomic Sequence ◽  
Critical Role ◽  
Protein Isoforms ◽  
Translation Initiation Site ◽  
Terminal Protein ◽  
Exon 2 ◽  
Protein 4.1R

Abstract Among the alternative pre-mRNA splicing events that characterize protein 4.1R gene expression, one involving exon 2′ plays a critical role in regulating translation initiation and N-terminal protein structure. Exon 2′ encompasses translation initiation site AUG1 and is located between alternative splice acceptor sites at the 5′ end of exon 2; its inclusion or exclusion from mature 4.1R mRNA regulates expression of longer or shorter isoforms of 4.1R protein, respectively. The current study reports unexpected complexity in the 5′ region of the 4.1R gene that directly affects alternative splicing of exon 2′. Identified far upstream of exon 2 in both mouse and human genomes were 3 mutually exclusive alternative 5′ exons, designated 1A, 1B, and 1C; all 3 are associated with strong transcriptional promoters in the flanking genomic sequence. Importantly, exons 1A and 1B splice differentially with respect to exon 2′, generating transcripts with different 5′ ends and distinct N-terminal protein coding capacity. Exon 1A-type transcripts splice so as to exclude exon 2′ and therefore utilize the downstream AUG2 for translation of 80-kDa 4.1R protein, whereas exon 1B transcripts include exon 2′ and initiate at AUG1 to synthesize 135-kDa isoforms. RNA blot analyses revealed that 1A transcripts increase in abundance in late erythroblasts, consistent with the previously demonstrated up-regulation of 80-kDa 4.1R during terminal erythroid differentiation. Together, these results suggest that synthesis of structurally distinct 4.1R protein isoforms in various cell types is regulated by a novel mechanism requiring coordination between upstream transcription initiation events and downstream alternative splicing events.

scholarly journals Novel Splice Site Mutation in the PROS1 Gene in a Polish Patient with Venous Thromboembolism: c.602-2delA, Splice Acceptor Site of Exon 7

Medicina ◽  
2020 ◽  
Vol 56 (9) ◽  
pp. 485
Author(s):  
Magdalena Mrożek ◽  
Ewa Wypasek ◽  
Martine Alhenc-Gelas ◽  
Daniel P. Potaczek ◽  
Anetta Undas
Keyword(s):  
Venous Thromboembolism ◽  
Sequence Analysis ◽  
Splice Site ◽  
Splice Site Mutation ◽  
Splice Acceptor Site ◽  
Acceptor Site ◽  
Nucleotide Position ◽  
Splice Acceptor ◽  
Site Mutation ◽  
History Of

We identified a novel splice site mutation of the PROS1 gene in a Polish family with protein S (PS) deficiency and explored the molecular pathogenesis of this previously undescribed variant. A novel mutation was detected in a 26-year-old woman with a history of venous thromboembolism (VTE) provoked by oral contraceptives. Her family history of VTE was positive. The sequence analysis of the PROS1 gene was performed in the proband and the proband’s family. The proband and their asymptomatic father had lower free PS levels (45% and 50%, respectively) and PS activity (48% and 44%, respectively). Total PS levels were normal (65.6% and 62.4%, respectively). The sequence analysis of the PROS1 gene revealed the presence of heterozygous deletion at the nucleotide position c.602-2 in intron 6, just upstream of exon 7, detected in the proband and her father. This variant alters the splice acceptor site of exon 7, and, according to the in silico prediction, it is highly likely to cause in-frame exon 7 skipping. We also presented follow-up data of two other Polish patients with PS deficiency associated with splice site mutations in PROS1 gene.

scholarly journals Activation of a Cryptic Splice Site of GFAP in a Patient With Adult-Onset Alexander Disease

2021 ◽  
Vol 7 (6) ◽  
pp. e626
Author(s):  
Eiichiro Amano ◽  
Tomokatsu Yoshida ◽  
Ikuko Mizuta ◽  
Jun Oyama ◽  
Shingo Sakashita ◽  
...  
Keyword(s):  
Splice Site ◽  
Late Onset ◽  
Splice Site Mutation ◽  
Alexander Disease ◽  
Splice Acceptor Site ◽  
Acceptor Site ◽  
Missense Mutations ◽  
Adult Onset ◽  
Splice Acceptor ◽  
Site Mutation

Background and ObjectiveAlexander disease (ALXDRD) is an autosomal dominant neurologic disorder caused by mutations in the glial fibrillary acidic protein (GFAP) gene and is pathologically defined by Rosenthal fiber accumulation. Most mutations are exonic missense mutations, and splice site mutations are rare. We report a very-late-onset autopsied case of adult-onset ALXDRD with a novel splice site mutation.MethodsGenetic testing of GFAP was performed by Sanger sequencing. Using autopsied brain tissues, GFAP transcript analysis was performed.ResultsThe patient presented mild upper motor neuron symptoms in contrast to the severe atrophy of spinal cord and medulla oblongata. The patient had c.619-1G>A mutation, which is located in the canonical splice acceptor site of intron 3. The brain RNA analysis identified the r.619_621del (p.Glu207del) mutation, which is explained by the activation of the cryptic splice acceptor site in the second and third nucleotides from the 5′ end of the exon 4.DiscussionGFAP gene expression analysis is necessary to clarify the effects of intronic mutations on splicing, even if they are in canonical splice sites. This case showed a much milder phenotype than those in previous cases with missense mutations at Glu207, thereby expanding the clinical spectrum of ALXDRD with Glu207 mutation.

Molecular Markers for Rapidly Identifying Candidate Genes in Chlamydomonas reinhardtii: ERY1 and ERY2 Encode Chloroplast Ribosomal Proteins

Genetics ◽  
2003 ◽  
Vol 164 (4) ◽  
pp. 1345-1353
Author(s):  
Amber K Bowers ◽  
Jennifer A Keller ◽  
Susan K Dutcher
Keyword(s):  
Molecular Markers ◽  
Chlamydomonas Reinhardtii ◽  
Candidate Genes ◽  
Splice Site ◽  
Genomic Dna ◽  
Ribosomal Proteins ◽  
Genomic Sequence ◽  
Point Mutations ◽  
Acceptor Site ◽  
Wild Type

Abstract To take advantage of available expressed sequence tags and genomic sequence, we have developed 64 PCR-based molecular markers in Chlamydomonas reinhardtii that map to the 17 linkage groups. These markers will allow the rapid association of a candidate gene sequence with previously identified mutations. As proof of principle, we have identified the genes encoded by the ERY1 and ERY2 loci. Mendelian mutations that confer resistance to erythromycin define three unlinked nuclear loci in C. reinhardtii. Candidate genes ribosomal protein L4 (RPL4) and L22 (RPL22) are tightly linked to the ERY1 locus and ERY2 locus, respectively. Genomic DNA for RPL4 from wild type and five mutant ery1 alleles was amplified and sequenced and three different point mutations were found. Two different glycine residues (G102 and G112) are replaced by aspartic acid and both are in the unstructured region of RPL4 that lines the peptide exit tunnel of the chloroplast ribosome. The other two alleles change a splice site acceptor site. Genomic DNA for RPL22 from wild type and three mutant ery2 alleles was amplified and sequenced and revealed three different point mutations. Two alleles have premature stop codons and one allele changes a splice site acceptor site.

scholarly journals Splice-acceptor site mutation in p53 gene of hu888 zebrafish line

2014 ◽  
Vol 56 (1) ◽  
pp. 115-121 ◽  
Author(s):  
Alicja Piasecka ◽  
Paweł Brzuzan ◽  
Maciej Woźny ◽  
Sławomir Ciesielski ◽  
Dariusz Kaczmarczyk
Keyword(s):  
P53 Gene ◽  
Splice Acceptor Site ◽  
Acceptor Site ◽  
Splice Acceptor ◽  
Site Mutation ◽  
Splice Acceptor Site Mutation

scholarly journals Complete Thyroxine-Binding Globulin (TBG) Deficiency Produced by a Mutation in Acceptor Splice Site Causing Frameshift and Early Termination of Translation (TBG-Kankakee)12

1998 ◽  
Vol 83 (10) ◽  
pp. 3604-3608
Author(s):  
Gisah A. Carvalho ◽  
Roy E. Weiss ◽  
Samuel Refetoff
Keyword(s):  
Splice Site ◽  
Messenger Rna ◽  
Restriction Site ◽  
Splice Junction ◽  
Early Termination ◽  
Coding Region ◽  
Acceptor Splice Site ◽  
Exon 2 ◽  
Gene Level ◽  
Exon 1

Fourteen T4-binding globulin (TBG) variants have been identified at the gene level. They are all located in the coding region of the gene and 6 produce complete deficiency of TBG (TBG-CD). We now describe the first mutation in a noncoding region producing TBG-CD. The proband was treated for over 20 yr with L-T4 because of fatigue associated with a low concentration of serum total T4. Fifteen family members were studied showing low total T4 inherited as an X chromosome-linked trait, and affected males had undetectable TBG in serum. Sequencing of the entire coding region and promoter of the TBG gene revealed no abnormality. However, an A to G transition was found in the acceptor splice junction of intron II that produced a new HaeIII restriction site cosegregating with the TBG-CD phenotype. Sequencing exon 1 to exon 3 of TBG complementary DNA reverse transcribed from messenger RNA of skin fibroblasts from an affected male, confirmed a shift in the ag acceptor splice site. This results in the insertion of a G in exon 2 and causes a frameshift and a premature stop at codon 195. This early termination of translation predicts a truncated TBG lacking 201 amino acids.

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