Faculty Opinions recommendation of The MicroRNA miR-124 promotes neuronal differentiation by triggering brain-specific alternative pre-mRNA splicing.

The Drosophila dopa decarboxylase gene, Ddc, is expressed in the hypoderm and in a small number of cells in the central nervous system (CNS). The unique Ddc primary transcript is alternatively spliced in these two tissues. We investigated whether Ddc splicing in the CNS is a general property of the CNS or a unique property of the cells that normally express Ddc by expressing the Ddc primary transcript ubiquitously under the control of an Hsp70 heat shock promoter. Under basal expression conditions, Ddc splicing shows normal tissue specificity, indicating that the regulation of Ddc splicing in the CNS is tissue specific rather than cell specific. Previous studies have shown that severe heat shock blocks mRNA splicing in cultured Drosophila melanogaster cells. Our results show that splicing of the heat shock-inducible Hsp83 transcript is very resistant to heat shock. In contrast, under either mild or severe heat shock, the splicing specificity of the heat shock-induced Ddc primary transcript is affected, leading to the accumulation of inappropriately high levels of the CNS splice form in non-CNS tissues. The chromosomal Ddc transcript is similarly affected. These results show unexpected heterogeneity in the splicing of individual mRNAs as a response to heat shock and suggest that the Ddc CNS-specific splicing pathway is the default.

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Tissue-specific alternative splicing of protein 4.1 inserts an exon necessary for formation of the ternary complex with erythrocyte spectrin and F-actin

Blood ◽

10.1182/blood.v82.8.2558.2558 ◽

1993 ◽

Vol 82 (8) ◽

pp. 2558-2563 ◽

Cited By ~ 57

Author(s):

WC Horne ◽

SC Huang ◽

PS Becker ◽

TK Tang ◽

EJ Jr Benz

Keyword(s):

Ternary Complex ◽

Mrna Splicing ◽

Actin Binding ◽

Protein 4.1 ◽

Alternative Mrna Splicing ◽

Actin Binding Domain ◽

Peripheral Membrane Protein ◽

Specific Alternative ◽

Erythroid Maturation ◽

Complete Protein

Abstract Erythrocyte protein 4.1 is an 78- to 80-Kd peripheral membrane protein that promotes the interaction of spectrin with actin protofilaments and links the resulting interlocking network to the integral membrane proteins. There are several isoforms of protein 4.1 that appear to be expressed in a restricted group of tissues. These arise from alternative mRNA splicing events that lead to the combinational insertion or deletion of at least 10 blocks of nucleotides (motifs) within the mature mRNA. One of these, motif I, consists of 63 nucleotides encoding 21 amino acids in the N-terminal region of the putative spectrin/actin-binding domain. The expression of the motif U- containing isoform occurs late in erythroid maturation. We generated recombinant isoforms of protein 4.1 and of the putative 10-Kd spectrin/actin-binding fragment that contain or lack this 21 amino acid sequence and examined their ability to form a ternary complex with erythrocyte spectrin and F-actin. The isoforms of the complete protein and of the 10-Kd fragment that contain the sequence encoded by motif I efficiently form the ternary complex. Isoforms that lack this sequence, but are otherwise identical, do not participate in the formation of the ternary complex. These results, in conjunction with the expression of motif I during late erythroid maturation, suggest that interaction with actin and the erythroid form of spectrin is a specialized property of the erythrocyte form of protein 4.1. Alternative mRNA splicing in developing red blood cells thus plays a key adaptive role in the formation of the highly specialized erythrocyte membrane.

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Expression and mRNA splicing of glycine receptor subunits and gephyrin during neuronal differentiation of P19 cells in vitro, studied by RT-PCR and immunocytochemistry

Developmental Brain Research ◽

10.1016/s0165-3806(96)00181-2 ◽

1997 ◽

Vol 98 (2) ◽

pp. 211-220 ◽

Cited By ~ 21

Author(s):

Susanne Heck ◽

Ralf Enz ◽

Christiane Richter-Landsberg ◽

Dietmar H. Blohm

Keyword(s):

Neuronal Differentiation ◽

Glycine Receptor ◽

Mrna Splicing ◽

P19 Cells ◽

Rt Pcr

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Tissue-specific alternative splicing of protein 4.1 inserts an exon necessary for formation of the ternary complex with erythrocyte spectrin and F-actin

Blood ◽

10.1182/blood.v82.8.2558.bloodjournal8282558 ◽

1993 ◽

Vol 82 (8) ◽

pp. 2558-2563 ◽

Cited By ~ 2

Author(s):

WC Horne ◽

SC Huang ◽

PS Becker ◽

TK Tang ◽

EJ Jr Benz

Keyword(s):

Ternary Complex ◽

Mrna Splicing ◽

Actin Binding ◽

Protein 4.1 ◽

Alternative Mrna Splicing ◽

Actin Binding Domain ◽

Peripheral Membrane Protein ◽

Specific Alternative ◽

Erythroid Maturation ◽

Complete Protein

Erythrocyte protein 4.1 is an 78- to 80-Kd peripheral membrane protein that promotes the interaction of spectrin with actin protofilaments and links the resulting interlocking network to the integral membrane proteins. There are several isoforms of protein 4.1 that appear to be expressed in a restricted group of tissues. These arise from alternative mRNA splicing events that lead to the combinational insertion or deletion of at least 10 blocks of nucleotides (motifs) within the mature mRNA. One of these, motif I, consists of 63 nucleotides encoding 21 amino acids in the N-terminal region of the putative spectrin/actin-binding domain. The expression of the motif U- containing isoform occurs late in erythroid maturation. We generated recombinant isoforms of protein 4.1 and of the putative 10-Kd spectrin/actin-binding fragment that contain or lack this 21 amino acid sequence and examined their ability to form a ternary complex with erythrocyte spectrin and F-actin. The isoforms of the complete protein and of the 10-Kd fragment that contain the sequence encoded by motif I efficiently form the ternary complex. Isoforms that lack this sequence, but are otherwise identical, do not participate in the formation of the ternary complex. These results, in conjunction with the expression of motif I during late erythroid maturation, suggest that interaction with actin and the erythroid form of spectrin is a specialized property of the erythrocyte form of protein 4.1. Alternative mRNA splicing in developing red blood cells thus plays a key adaptive role in the formation of the highly specialized erythrocyte membrane.

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Expression changes confirm genomic variants predicted to result in allele-specific, alternative mRNA splicing

10.1101/549089 ◽

2019 ◽

Cited By ~ 2

Author(s):

Eliseos J. Mucaki ◽

Ben C. Shirley ◽

Peter K. Rogan

Keyword(s):

Gene Expression ◽

Information Theory ◽

Splice Site ◽

Mrna Splicing ◽

Splice Sites ◽

Rt Pcr ◽

Alternative Mrna Splicing ◽

Site Use ◽

Allele Specific ◽

Specific Alternative

AbstractSplice isoform structure and abundance can be affected by either non-coding or masquerading coding variants that alter the structure or abundance of transcripts. When these variants are common in the population, these non-constitutive transcripts are sufficiently frequent so as to resemble naturally occurring, alternative mRNA splicing. Prediction of the effects of such variants has been shown to be accurate using information theory-based methods. Single nucleotide polymorphisms (SNPs) predicted to significantly alter natural and/or cryptic splice site strength were shown to affect gene expression. Splicing changes for known SNP genotypes were confirmed in HapMap lymphoblastoid cell lines with gene expression microarrays and custom designed q-RT-PCR or TaqMan assays. The majority of these SNPs (15 of 22) as well as an independent set of 24 variants were then subjected to RNAseq analysis using the ValidSpliceMut web beacon (http://validsplicemut.cytognomix.com), which is based on data from the Cancer Genome Atlas and International Cancer Genome Consortium. SNPs from different genes analyzed with gene expression microarray and q-RT-PCR exhibited significant changes in affected splice site use. Thirteen SNPs directly affected exon inclusion and 10 altered cryptic site use. Homozygous SNP genotypes resulting in stronger splice sites exhibited higher levels of processed mRNA than alleles associated with weaker sites. Four SNPs exhibited variable expression among individuals with the same genotypes, masking statistically significant expression differences between alleles. Genome-wide information theory and expression analyses (RNAseq) in tumour exomes and genomes confirmed splicing effects for 7 of the HapMap SNP and 14 SNPs identified from tumour genomes. q-RT-PCR resolved rare splice isoforms with read abundance too low for statistical significance in ValidSpliceMut. Nevertheless, the web-beacon provides evidence of unanticipated splicing outcomes, for example, intron retention due to compromised recognition of constitutive splice sites. Thus, ValidSpliceMut and q-RT-PCR represent complementary resources for identification of allele-specific, alternative splicing.

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