The effects of defective Suppressor-mutator insertions on the expression of a Bronze-1 allele in maize

Genome ◽  
1989 ◽  
Vol 31 (2) ◽  
pp. 980-986
Author(s):  
Hwa Yeong Kim ◽  
Victor Raboy ◽  
John W. Schiefelbein ◽  
Oliver E. Nelson
Keyword(s):  
Enzymatic Activity ◽  
Transposable Element ◽  
Splice Site ◽  
Splice Sites ◽  
Base Pairs ◽  
Acceptor Splice Site ◽  
Biochemical Analyses ◽  
Bronze Locus ◽  
Maize Transposable Element ◽  
Mutator Element

Molecular and biochemical analyses of the maize transposable element mutant bronze-m13, which resulted from the insertion of a defective Suppressor-mutator element in an exon of a Bronze-1 allele, and of changes of state derived from bronze-m13 by internal deletions within the element have revealed how these mutant alleles condition a nonmutant phenotype in the absence of a trans-active Suppressor-mutator. The transposable element insertions are all in the same position, 38 base pairs 3′ to the single intron present in the bz locus. The insertions are transcribed with the gene, and the pre-mRNAs of bronze-m13 and CS1, CS5, CS6, and CS12 are then spliced using the intron donor splice site and either one of two acceptor splice sites in the defective Suppressor-mutator element. Only one of these two messages is translated to yield a functional enzyme. The pre-mRNA of CS9 is spliced only in the reaction that gives a translatable message since the pre-mRNA lacks the alternate acceptor splice site. The splicing reactions are detailed and related to the very different amounts of enzymatic activity produced by these alleles. The presence of an antisense message in CS12 plants to the defective Suppressor-mutator sequence transcribed with the bronze locus is also discussed.Key words: maize transposable element, Suppressor-mutator, splicing.

scholarly journals Frequency Analysis of the Splice Site Regions in Different Organisms

2007 ◽  
Vol 4 (2) ◽  
pp. 24-46 ◽  
Author(s):  
T. Shashi Rekha ◽  
Chanchal K Mitra
Keyword(s):  
Comparative Analysis ◽  
Frequency Analysis ◽  
Splice Site ◽  
Splice Sites ◽  
Acceptor Splice Site ◽  
Donor Region ◽  
Donor And Acceptor ◽  
Donor Acceptor ◽  
Score Pattern ◽  
Degree Of Similarity

Summary We have carried out a comparative analysis of the sub-sequences of size six| ten at the (donor| acceptor) splice site regions of five different organisms. The frequency analysis of the unique sub-sequences at the donor and acceptor regions suggests that the distribution of their occurrence is approximately exponential. We have observed that the number of unique sub-sequences (occurring with different frequencies) at the donor region are less than at the acceptor, suggesting that the sub-sequences at the acceptor region are more variable. The sub-sequences with high percentage of occurrence (uniqueness) are considered to be highly involved in splicing. Our analysis suggests that sub-sequences of length ~6-8 nucleotides (nt) at the splice sites – with six bases in intron (including the two central, conserved dinucleotides) and two bases in exon are optimal for the efficient assembly and binding of the spliceosomal complex during the process of splicing. The score pattern obtained by the alignment of the nucleotides at the donor region with the acceptor and vice-versa also suggests that a single sub-sequence at the donor region have different degree of similarity with sub-sequences at the acceptor thus determining that the donor sub-sequences are more crucial in pairing with the corresponding acceptor sub-sequences during the process of splicing.

scholarly journals The effect of disease-associated HRPT2 mutations on splicing

2009 ◽  
Vol 201 (3) ◽  
pp. 387-396 ◽  
Author(s):  
Michael A Hahn ◽  
Julie McDonnell ◽  
Deborah J Marsh
Keyword(s):  
Splice Site ◽  
Hot Spot ◽  
Splice Sites ◽  
Loss Of Function ◽  
Splice Enhancer ◽  
Aberrant Splicing ◽  
Acceptor Splice Site ◽  
Web Based ◽  
Exonic Splice

Mutations in the tumour suppressor HRPT2 occur in patients with parathyroid carcinoma, kidney tumours and Hyperparathyroidism–Jaw Tumour syndrome. Disruption of exonic splicing through mutation of donor/acceptor splice sites or exonic splice enhancer (ESE) sites leads to loss of function of a number of major tumour suppressors including BRCA1, APC and MLH1. Given that the effect of HRPT2 mutations on splicing has not been widely studied, we used an in vitro splicing assay to determine whether 17 HRPT2 mutations located in hot-spot and other exons predicted to disrupt ESE consensus sites led to aberrant splicing. Using two independent web-based prediction programs, the majority of these mutations were predicted to disrupt ESE consensus sites; however, aberrant splicing of HRPT2 transcripts was not observed. Canonical donor or acceptor splice site mutations were also investigated using this splicing assay and transcripts assessed from tumour tissue. Splice site mutations were shown to lead to either exon skipping or retention of intronic sequences through the use of cryptic splice sites comprised of non-classical splicing signals. Aberrant splicing caused by disruption of ESE sites does not appear to have a major role in HRPT2-associated disease; however, premature truncation of parafibromin as the result of canonical donor or acceptor splice site mutations is associated with pathogenicity. Functional splicing assays must be undertaken in order to confirm web-based software predictions of the modification of putative ESE sites by disease-associated mutations.

scholarly journals The upstream 5′ splice site remains associated to the transcription machinery during intron synthesis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yodfat Leader ◽  
Galit Lev Maor ◽  
Matan Sorek ◽  
Ronna Shayevitch ◽  
Maram Hussein ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Splice Site ◽  
Human Cells ◽  
Splice Sites ◽  
Base Pairs ◽  
U1 Snrna ◽  
U1 Snrnp ◽  
Bona Fide ◽  
Rna Polymerase Ii Transcription

AbstractIn the earliest step of spliceosome assembly, the two splice sites flanking an intron are brought into proximity by U1 snRNP and U2AF along with other proteins. The mechanism that facilitates this intron looping is poorly understood. Using a CRISPR interference-based approach to halt RNA polymerase II transcription in the middle of introns in human cells, we discovered that the nascent 5′ splice site base pairs with a U1 snRNA that is tethered to RNA polymerase II during intron synthesis. This association functionally corresponds with splicing outcome, involves bona fide 5′ splice sites and cryptic intronic sites, and occurs transcriptome-wide. Overall, our findings reveal that the upstream 5′ splice sites remain attached to the transcriptional machinery during intron synthesis and are thus brought into proximity of the 3′ splice sites; potentially mediating the rapid splicing of long introns.

scholarly journals A novel CHD7 variant disrupting acceptor splice site in a patient with mild features of CHARGE syndrome: a case report

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Evelina Siavrienė ◽  
Gunda Petraitytė ◽  
Violeta Mikštienė ◽  
Tautvydas Rančelis ◽  
Živilė Maldžienė ◽  
...  
Keyword(s):  
Case Report ◽  
Splice Site ◽  
Charge Syndrome ◽  
Acceptor Splice Site

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.

Nuclear pre-mRNA processing in plants: distinct modes of 3'-splice-site selection in plants and animals

1988 ◽  
Vol 8 (5) ◽  
pp. 2042-2051
Author(s):  
K Wiebauer ◽  
J J Herrero ◽  
W Filipowicz
Keyword(s):  
Splice Site ◽  
Hela Cells ◽  
Site Selection ◽  
Human Growth ◽  
Mrna Processing ◽  
Beta Globin ◽  
Splice Sites ◽  
Splice Site Selection ◽  
Consensus Sequences ◽  
Intron 2

The report that human growth hormone pre-mRNA is not processed in transgenic plant tissues (A. Barta, K. Sommergruber, D. Thompson, K. Hartmuth, M.A. Matzke, and A.J.M. Matzke, Plant Mol. Biol. 6:347-357, 1986) has suggested that differences in mRNA splicing processes exist between plants and animals. To gain more information about the specificity of plant pre-mRNA processing, we have compared the splicing of the soybean leghemoglobin pre-mRNA with that of the human beta-globin pre-mRNA in transfected plant (Orychophragmus violaceus and Nicotiana tabacum) protoplasts and mammalian (HeLa) cells. Of the three introns of leghemoglobin pre-mRNA, only intron 2 was correctly and efficiently processed in HeLa cells. The 5' splice sites of the remaining two introns were faithfully recognized, but correct processing of the 3' sites took place only rarely (intron 1) or not at all (intron 3); cryptic 3' splice sites were used instead. While the first intron in human beta-globin pre-mRNA was not spliced in transfected plant protoplasts, intron 2 processing occurred at a low level, indicating that some mammalian introns can be recognized by the plant intron-splicing machinery. However, excision of intron 2 proved to be incorrect, involving the authentic 5' splice site and a cryptic 3' splice site. Our results indicate that the mechanism of 3'-splice-site selection during intron excision differs between plants and animals. This conclusion is supported by analysis of the 3'-splice-site consensus sequences in animal and plant introns which revealed that polypyrimidine tracts, characteristic of animal introns, are not present in plant pre-mRNAs. It is proposed that an elevated AU content of plant introns is important for their processing.

Control of adenovirus E1B mRNA synthesis by a shift in the activities of RNA splice sites

1984 ◽  
Vol 4 (5) ◽  
pp. 966-972
Author(s):  
C Montell ◽  
E F Fisher ◽  
M H Caruthers ◽  
A J Berk
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Early Phase ◽  
Late Phase ◽  
Productive Infection ◽  
Splice Sites ◽  
Mrna Synthesis ◽  
Adenovirus E1b ◽  
Cryptic Splice Sites ◽  
Rna Splice Sites

The primary transcript from adenovirus 2 early region 1B (E1B) is processed by differential RNA splicing into two overlapping mRNAs, 13S and 22S. The 22S mRNA is the major E1B mRNA during the early phase of infection, whereas the 13S mRNA predominates during the late phase. In previous work, it has been shown that this shift in proportions of the E1B mRNAs is influenced by increased cytoplasmic stability of the 13S mRNA at late times in infection. Two observations presented here demonstrate that the increase in proportion of the 13S mRNA at late times is also regulated by a change in the specificity of RNA splicing. First, the relative concentrations of the 13S to 22S nuclear RNAs were not constant throughout infection but increased at late times. Secondly, studies with the mutant, adenovirus 2 pm2250 , provided evidence that there was an increased propensity to utilize a 5' splice in the region of the 13S 5' splice site at late times in infection. Adenovirus 2 pm2250 has a G----C transversion in the first base of E1B 13S mRNA intron preventing splicing of the 13S mRNA but not of the 22S mRNA. During the early phase of a pm2250 infection, the E1B primary transcripts were processed into the 22S mRNA only. However, during the late phase, when the 13S mRNA normally predominates, E1B primary transcripts were also processed by RNA splicing at two formerly unused or cryptic 5' splice sites. Both cryptic splice sites were located much closer to the disrupted 13S 5' splice site than to the 22S 5' splice site. Thus, the temporal increase in proportion of the 13S mRNA to the 22S mRNA is regulated by two processes, an increase in cytoplasmic stability of the 13S mRNA and an increased propensity to utilize the 13S 5' splice site during the late phase of infection. Adenovirus 2 pm2250 was not defective for productive infection of HeLa cells or for transformation of rat cells.

A novel protein factor is required for use of distal alternative 5' splice sites in vitro

1991 ◽  
Vol 11 (12) ◽  
pp. 5945-5953
Author(s):  
J E Harper ◽  
J L Manley
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Deae Cellulose ◽  
Simian Virus 40 ◽  
Nuclear Extract ◽  
Simian Virus ◽  
Splice Sites ◽  
Splice Site Selection ◽  
Small Nuclear Ribonucleoprotein

Adenovirus E1A pre-mRNA was used as a model to examine alternative 5' splice site selection during in vitro splicing reactions. Strong preference for the downstream 13S 5' splice site over the upstream 12S or 9S 5' splice sites was observed. However, the 12S 5' splice site was used efficiently when a mutant pre-mRNA lacking the 13S 5' splice site was processed, and 12S splicing from this substrate was not reduced by 13S splicing from a separate pre-mRNA, demonstrating that 13S splicing reduced 12S 5' splice site selection through a bona fide cis-competition. DEAE-cellulose chromatography of nuclear extract yielded two fractions with different splicing activities. The bound fraction contained all components required for efficient splicing of simple substrates but was unable to utilize alternative 5' splice sites. In contrast, the flow-through fraction, which by itself was inactive, contained an activity required for alternative splicing and was shown to stimulate 12S and 9S splicing, while reducing 13S splicing, when added to reactions carried out by the bound fraction. Furthermore, the activity, which we have called distal splicing factor (DSF), enhanced utilization of an upstream 5' splice site on a simian virus 40 early pre-mRNA, suggesting that the factor acts in a position-dependent, substrate-independent fashion. Several lines of evidence are presented suggesting that DSF is a non-small nuclear ribonucleoprotein protein. Finally, we describe a functional interaction between DSF and ASF, a protein that enhances use of downstream 5' splice sites.

scholarly journals U1 small nuclear RNAs with altered specificity can be stably expressed in mammalian cells and promote permanent changes in pre-mRNA splicing.

1993 ◽  
Vol 13 (5) ◽  
pp. 2666-2676 ◽  
Author(s):  
J B Cohen ◽  
S D Broz ◽  
A D Levinson
Keyword(s):  
Splice Site ◽  
Mammalian Cells ◽  
Mrna Processing ◽  
Mrna Splicing ◽  
Small Nuclear Rna ◽  
Splice Sites ◽  
Gene Complementation ◽  
Small Nuclear Rnas ◽  
Nuclear Rna ◽  
Mutant Genes

Pre-mRNA 5' splice site activity depends, at least in part, on base complementarity to U1 small nuclear RNA. In transient coexpression assays, defective 5' splice sites can regain activity in the presence of U1 carrying compensatory changes, but it is unclear whether such mutant U1 RNAs can be permanently expressed in mammalian cells. We have explored this issue to determine whether U1 small nuclear RNAs with altered specificity may be of value to rescue targeted mutant genes or alter pre-mRNA processing profiles. This effort was initiated following our observation that U1 with specificity for a splice site associated with an alternative H-ras exon substantially reduced the synthesis of the potentially oncogenic p21ras protein in transient assays. We describe the development of a mammalian complementation system that selects for removal of a splicing-defective intron placed within a drug resistance gene. Complementation was observed in proportion to the degree of complementarity between transfected mutant U1 genes and different defective splice sites, and all cells selected in this manner were found to express mutant U1 RNA. In addition, these cells showed specific activation of defective splice sites presented by an unlinked reporter gene. We discuss the prospects of this approach to permanently alter the expression of targeted genes in mammalian cells.

scholarly journals Mutations in Transglutaminase 1 Gene in Autosomal Recessive Congenital Ichthyosis in Egyptian Families

2004 ◽  
Vol 20 (6) ◽  
pp. 325-332 ◽  
Author(s):  
R. M. Shawky ◽  
N.S. Sayed ◽  
N.A. Elhawary
Keyword(s):  
Restriction Endonuclease ◽  
Splice Site ◽  
Autosomal Recessive ◽  
Splice Mutation ◽  
The Other ◽  
Splice Acceptor ◽  
Acceptor Splice Site ◽  
Congenital Ichthyosis ◽  
Transglutaminase 1 ◽  
Autosomal Recessive Congenital Ichthyosis

Autosomal recessive congenital ichthyosis (ARCI) is a rare heterogeneous keratinization disorder of the skin. It is clinically divided into 2 subtypes, lamellar ichthyosis (LI) and congenital ichthyosiformis erythroderma (CIE). We investigated forty-three ARCI Egyptian individuals in 16 severe LI, and 10 CIE families. We identified 5 alleles in two Egyptian families as having intron-5/exon-6 splice acceptor mutation recognized by theMspIrestriction endonuclease. This promoted to a frequency of 9.6% for this mutation (5 splice-mutation alleles/52 alleles tested). We extended our previous dataset to update the detection of R142H mutation in 4 CIE Egyptian families and one LI phenotype (frequency of 28.8%; 15/52), whereas we still had no R141H among our Egyptian population. There was no correlation between phenotype and genotype in our study. Surprisingly, the mutant alleles detected in intron-5 acceptor splice-site were associated with the other extreme of CIE phenotypes rather than the severe LI form. We clearly demonstrated that the ARCI Egyptian families in Upper Egypt was ethnically pure and had a tendency not to be a hybrid with other populations in Lower Egypt, Delta zone and Cairo city.

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