scholarly journals Alternative 5' splice site selection induced by heat shock.

1994 ◽  
Vol 14 (1) ◽  
pp. 567-575 ◽  
Author(s):  
H Takechi ◽  
N Hosokawa ◽  
K Hirayoshi ◽  
K Nagata
Keyword(s):  
Alternative Splicing ◽  
Heat Shock ◽  
Site Selection ◽  
Donor Site ◽  
Splice Donor Site ◽  
In Vivo Model ◽  
Recognition Mechanism ◽  
Amino Acid Analog ◽  
Alternatively Spliced

The mouse HSP47 gene consists of six exons separated by five introns. Three HSP47 cDNAs differing only in their 5' noncoding regions have been reported. One of these alternatively spliced mRNAs was detected only after heat shock, which caused an alternative 5' splice donor site selection. Other stress inducers, including an amino acid analog and sodium arsenite, had no effect on the alternative splicing. The alternatively spliced mRNA, which was 169 nucleotides longer in the 5' noncoding region compared to mRNA transcribed in non-heat shock conditions, was efficiently translated under heat shock conditions. This novel finding that alternative splicing is caused by artificial treatment like heat shock will provide a useful in vivo model for understanding the exon-intron recognition mechanism as well as heat shock-induced alterations in gene expression.

Alternative 5' splice site selection induced by heat shock

1994 ◽  
Vol 14 (1) ◽  
pp. 567-575
Author(s):  
H Takechi ◽  
N Hosokawa ◽  
K Hirayoshi ◽  
K Nagata
Keyword(s):  
Alternative Splicing ◽  
Heat Shock ◽  
Site Selection ◽  
Donor Site ◽  
Splice Donor Site ◽  
In Vivo Model ◽  
Recognition Mechanism ◽  
Amino Acid Analog ◽  
Alternatively Spliced

The mouse HSP47 gene consists of six exons separated by five introns. Three HSP47 cDNAs differing only in their 5' noncoding regions have been reported. One of these alternatively spliced mRNAs was detected only after heat shock, which caused an alternative 5' splice donor site selection. Other stress inducers, including an amino acid analog and sodium arsenite, had no effect on the alternative splicing. The alternatively spliced mRNA, which was 169 nucleotides longer in the 5' noncoding region compared to mRNA transcribed in non-heat shock conditions, was efficiently translated under heat shock conditions. This novel finding that alternative splicing is caused by artificial treatment like heat shock will provide a useful in vivo model for understanding the exon-intron recognition mechanism as well as heat shock-induced alterations in gene expression.

Exon truncation by alternative splicing of murine ICAM-1

2002 ◽  
Vol 12 (1) ◽  
pp. 47-51 ◽  
Author(s):  
Joseph P. Mizgerd ◽  
Matt R. Spieker ◽  
Michal M. Lupa
Keyword(s):  
Alternative Splicing ◽  
Splice Variant ◽  
Donor Site ◽  
Exon Skipping ◽  
Intercellular Adhesion Molecule ◽  
Splice Donor Site ◽  
Intercellular Adhesion Molecule 1 ◽  
Membrane Expression ◽  
Alternatively Spliced ◽  
Ig Domain

The murine gene for intercellular adhesion molecule-1 (ICAM-1) encodes multiple products, arising from alternative splicing. Full-length ICAM-1 contains five extracellular Ig domains, each encoded by a separate exon. Alternatively spliced forms have Ig domains 2, 3, and/or 4 excised as a result of exon skipping. We report here a novel splice variant of murine ICAM-1, resulting from exon truncation rather than exon skipping and affecting Ig domain 5. A 5′ splice donor site within exon 6 generates transcripts missing 69 nucleic acids from the 3′ terminus of the exon. This in-frame exon truncation is predicted to replace 24 amino acids within Ig domain 5 with a single aspartic acid residue, yielding a structure other than an Ig domain immediately external to the membrane. Expression of this alternatively spliced form is induced in mouse lungs, spleen, and kidneys during LPS-induced pulmonary inflammation. Since the affected region is critical for ICAM-1 presentation, dimerization, and solubilization, this alternative splice variant may have unique physiological functions.

scholarly journals Comparison of in vitro and in vivo splice site selection in kappa-immunoglobulin precursor mRNA.

1988 ◽  
Vol 8 (6) ◽  
pp. 2610-2619 ◽  
Author(s):  
D E Lowery ◽  
B G Van Ness
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Donor Site ◽  
Splice Donor Site ◽  
Splice Sites ◽  
Splice Donor ◽  
Splice Site Selection ◽  
Nuclear Extracts

The processing of a number of kappa-immunoglobulin primary mRNA (pre-mRNA) constructs has been examined both in vitro and in vivo. When a kappa-immunoglobulin pre-mRNA containing multiple J segment splice sites is processed in vitro, the splice sites are used with equal frequency. The presence of signal exon, S-V intron, or variable (V) region has no effect on splice site selection in vitro. Nuclear extracts prepared from a lymphoid cell line do not restore correct splice site selection. Splice site selection in vitro can be altered by changing the position or sequence of J splice donor sites. These results differ from the processing of similar pre-mRNAs expressed in vivo by transient transfection. The 5'-most J splice donor site was exclusively selected in vivo, even in nonlymphoid cells, and even in transcripts where in vitro splicing favored a 3' J splice site. The in vitro results are consistent with a model proposing that splice site selection is influenced by splice site strength and proximity; however, our in vivo results demonstrate a number of discrepancies with such a model and suggest that splice site selection may be coupled to transcription or a higher-order nuclear structure.

scholarly journals A novel pathway for alternative splicing: identification of an RNA intermediate that generates an alternative 5' splice donor site not present in the primary transcript of AMPD1.

1990 ◽  
Vol 10 (10) ◽  
pp. 5271-5278 ◽  
Author(s):  
I Mineo ◽  
P R Clarke ◽  
R L Sabina ◽  
E W Holmes
Keyword(s):  
Alternative Splicing ◽  
Donor Site ◽  
Specific Pattern ◽  
Splice Donor Site ◽  
Splice Donor ◽  
Primary Transcript ◽  
Exon 2 ◽  
Tissue Specific ◽  
Exon 1 ◽  
Alternatively Spliced

AMP deaminase (AMPD) is a central enzyme in eucaryotic energy metabolism, and tissue-specific as well as stage-specific isoforms are found in many vertebrates. This study demonstrates the AMPD1 gene product in rat is alternatively spliced. The second exon, a 12-base miniexon, was found to be excluded or included in a tissue-specific and stage-specific pattern. This example of cassette splicing utilizes a unique pathway through an RNA intermediate that generates an alternative 5' splice donor site at the point where exon 2 is ligated to exon 1. In the analogous intermediate of human AMPD1, the potential 5' splice donor site created at the boundary of exon 1 and exon 2 was a poor substrate for splicing because of differences in exon 2 sequences, and human AMPD1 was not alternatively spliced. These results demonstrate that in some cases alternative splicing may proceed through an RNA intermediate that generates an alternative splice donor site not present in the primary transcript. Discrimination between alternative 5' splice donor sites in the RNA intermediate of AMPD1 is apparently controlled by tissue-specific and stage-specific signals.

Comparison of in vitro and in vivo splice site selection in kappa-immunoglobulin precursor mRNA

1988 ◽  
Vol 8 (6) ◽  
pp. 2610-2619
Author(s):  
D E Lowery ◽  
B G Van Ness
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Donor Site ◽  
Splice Donor Site ◽  
Splice Sites ◽  
Splice Donor ◽  
Splice Site Selection ◽  
Nuclear Extracts

The processing of a number of kappa-immunoglobulin primary mRNA (pre-mRNA) constructs has been examined both in vitro and in vivo. When a kappa-immunoglobulin pre-mRNA containing multiple J segment splice sites is processed in vitro, the splice sites are used with equal frequency. The presence of signal exon, S-V intron, or variable (V) region has no effect on splice site selection in vitro. Nuclear extracts prepared from a lymphoid cell line do not restore correct splice site selection. Splice site selection in vitro can be altered by changing the position or sequence of J splice donor sites. These results differ from the processing of similar pre-mRNAs expressed in vivo by transient transfection. The 5'-most J splice donor site was exclusively selected in vivo, even in nonlymphoid cells, and even in transcripts where in vitro splicing favored a 3' J splice site. The in vitro results are consistent with a model proposing that splice site selection is influenced by splice site strength and proximity; however, our in vivo results demonstrate a number of discrepancies with such a model and suggest that splice site selection may be coupled to transcription or a higher-order nuclear structure.

A novel pathway for alternative splicing: identification of an RNA intermediate that generates an alternative 5' splice donor site not present in the primary transcript of AMPD1

1990 ◽  
Vol 10 (10) ◽  
pp. 5271-5278
Author(s):  
I Mineo ◽  
P R Clarke ◽  
R L Sabina ◽  
E W Holmes
Keyword(s):  
Alternative Splicing ◽  
Donor Site ◽  
Specific Pattern ◽  
Splice Donor Site ◽  
Splice Donor ◽  
Primary Transcript ◽  
Exon 2 ◽  
Tissue Specific ◽  
Exon 1 ◽  
Alternatively Spliced

AMP deaminase (AMPD) is a central enzyme in eucaryotic energy metabolism, and tissue-specific as well as stage-specific isoforms are found in many vertebrates. This study demonstrates the AMPD1 gene product in rat is alternatively spliced. The second exon, a 12-base miniexon, was found to be excluded or included in a tissue-specific and stage-specific pattern. This example of cassette splicing utilizes a unique pathway through an RNA intermediate that generates an alternative 5' splice donor site at the point where exon 2 is ligated to exon 1. In the analogous intermediate of human AMPD1, the potential 5' splice donor site created at the boundary of exon 1 and exon 2 was a poor substrate for splicing because of differences in exon 2 sequences, and human AMPD1 was not alternatively spliced. These results demonstrate that in some cases alternative splicing may proceed through an RNA intermediate that generates an alternative splice donor site not present in the primary transcript. Discrimination between alternative 5' splice donor sites in the RNA intermediate of AMPD1 is apparently controlled by tissue-specific and stage-specific signals.

scholarly journals Expression of novel isoforms of carnitine palmitoyltransferase I (CPT-1) generated by alternative splicing of the CPT-Iβ gene

1998 ◽  
Vol 334 (1) ◽  
pp. 225-231 ◽  
Author(s):  
Geng-Sheng YU ◽  
Yi-Chun LU ◽  
Tod GULICK
Keyword(s):  
Alternative Splicing ◽  
Donor Site ◽  
Carnitine Palmitoyltransferase ◽  
Membrane Topology ◽  
Splice Donor Site ◽  
The Novel ◽  
Pcr Screening ◽  
Mitochondrial Fatty Acid ◽  
Carnitine Palmitoyltransferase I ◽  
Cpt I

Carnitine palmitoyltransferase I (CPT-I) catalyses the rate-determining step in mitochondrial fatty acid β-oxidation. The enzyme has two cognate structural genes that are preferentially expressed in liver (α) or fat and muscle (β). We hypothesized the existence of additional isoforms in heart to account for unique kinetic characteristics of enzyme activity in this tissue. Hybridization and PCR screening of a human cardiac cDNA library revealed the expression of two novel CPT-I isoforms generated by alternative splicing of the CPT-Iβ transcript, in addition to the β and α cDNA species previously described. Ribonuclease protection and reverse transcriptase-mediated PCR assays confirmed the presence of mRNA species of each splicing variant in heart, skeletal muscle and liver, with differing relative concentrations in the tissues. The novel splicing variants omit exons or utilize a cryptic splice donor site within an exon. Deduced polypeptide sequences of the novel enzymes include omissions in the region of putative membrane-spanning and malonyl-CoA regulatory domains compared with the previously described CPT-Is, implying that the encoded enzymes will exhibit unique features with respect to outer mitochondrial membrane topology and response to physiological and pharmacological inhibitors.

scholarly journals Induction of Heat Shock Protein 70 in Mouse RPE as an In Vivo Model of Transpupillary Thermal Stimulation

2020 ◽  
Vol 21 (6) ◽  
pp. 2063
Author(s):  
Mooud Amirkavei ◽  
Marja Pitkänen ◽  
Ossi Kaikkonen ◽  
Kai Kaarniranta ◽  
Helder André ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Response ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
In Vivo Model ◽  
Tunel Staining ◽  
Long Duration ◽  
Shock Response

The induction of heat shock response in the macula has been proposed as a useful therapeutic strategy for retinal neurodegenerative diseases by promoting proteostasis and enhancing protective chaperone mechanisms. We applied transpupillary 1064 nm long-duration laser heating to the mouse (C57Bl/6J) fundus to examine the heat shock response in vivo. The intensity and spatial distribution of heat shock protein (HSP) 70 expression along with the concomitant probability for damage were measured 24 h after laser irradiation in the mouse retinal pigment epithelium (RPE) as a function of laser power. Our results show that the range of heating powers for producing heat shock response while avoiding damage in the mouse RPE is narrow. At powers of 64 and 70 mW, HSP70 immunostaining indicates 90 and 100% probability for clearly elevated HSP expression while the corresponding probability for damage is 20 and 33%, respectively. Tunel staining identified the apoptotic regions, and the estimated 50% damaging threshold probability for the heating (ED50) was ~72 mW. The staining with Bestrophin1 (BEST1) demonstrated RPE cell atrophy with the most intense powers. Consequently, fundus heating with a long-duration laser provides an approachable method to develop heat shock-based therapies for the RPE of retinal disease model mice.

scholarly journals Influence of Intron Length on Alternative Splicing of CD44

1998 ◽  
Vol 18 (10) ◽  
pp. 5930-5941 ◽  
Author(s):  
Martyn V. Bell ◽  
Alison E. Cowper ◽  
Marie-Paule Lefranc ◽  
John I. Bell ◽  
Gavin R. Screaton
Keyword(s):  
Alternative Splicing ◽  
Genomic Structure ◽  
Single Gene ◽  
Intron Length ◽  
Protein Isoforms ◽  
Major Influence ◽  
Eukaryotic Genes ◽  
Alternatively Spliced ◽  
Alternatively Spliced Exons

ABSTRACT Although the splicing of transcripts from most eukaryotic genes occurs in a constitutive fashion, some genes can undergo a process of alternative splicing. This is a genetically economical process which allows a single gene to give rise to several protein isoforms by the inclusion or exclusion of sequences into or from the mature mRNA. CD44 provides a unique example; more than 1,000 possible isoforms can be produced by the inclusion or exclusion of a central tandem array of 10 alternatively spliced exons. Certain alternatively spliced exons have been ascribed specific functions; however, independent regulation of the inclusion or skipping of each of these exons would clearly demand an extremely complex regulatory network. Such a network would involve the interaction of many exon-specific trans-acting factors with the pre-mRNA. Therefore, to assess whether the exons are indeed independently regulated, we have examined the alternative exon content of a large number of individual CD44 cDNA isoforms. This analysis shows that the downstream alternatively spliced exons are favored over those lying upstream and that alternative exons are often included in blocks rather than singly. Using a novel in vivo alternative splicing assay, we show that intron length has a major influence upon the alternative splicing of CD44. We propose a kinetic model in which short introns may overcome the poor recognition of alternatively spliced exons. These observations suggest that for CD44, intron length has been exploited in the evolution of the genomic structure to enable tissue-specific patterns of splicing to be maintained.

Ott1(Rbm15) Regulates Thpo Response In HSCs Through Epigenetic Modifications Directing Alternative Splicing Of C-Mpl

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 222-222
Author(s):  
Nan Xiao ◽  
Kayla Morlock ◽  
Jonathan L Jesneck ◽  
Glen D Raffel
Keyword(s):  
Bone Marrow ◽  
Alternative Splicing ◽  
Histone Acetylation ◽  
Epigenetic Modifications ◽  
Class I ◽  
Fusion Partner ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Alternatively Spliced

Abstract Thrombopoietin (Thpo), through its receptor c-Mpl, is essential for Hematopoietic Stem Cell (HSC) function and has a dose-dependent effect in which low concentrations promote quiescence and self-renewal in contrast to high Thpo concentrations which promote proliferation. Thpo production is largely stable in vivo, therefore it is unclear how this dual response is evoked physiologically. HSCs deleted for c-Mpl are unable to tolerate proliferative stress. Ott1(Rbm15), the 5’ fusion partner in t(1;22) acute megakaryocytic leukemia, is also essential for maintaining HSC quiescence during proliferative stress, however the mechanism has not been elucidated. Total c-Mpl expression in Ott1-deleted HSCs does not significantly differ from wild type, however, the existence of a cross-species, conserved isoform, Mpl-TR, with dominant negative activity, suggests a potential mechanism for affecting c-Mpl signaling via alternative splicing. Ott1 is a spliceosome component, is implicated in RNA processing and possesses RNA Recognition Motifs, yet has not been linked with any known physiologic targets. Analysis of c-Mpl isoforms in HSC-containing Lin-Sca1+c-Kit+ fractions and fetal liver megakaryocytes showed a marked increase in the ratio of Mpl-TR transcript. Ott1-deleted HSC populations displayed reduced Stat5 phosphorylation in response to Thpo stimulation consistent with decreased Mpl signaling. Exogenous expression of Mpl-TR in wild type bone marrow dramatically reduced short and long term engraftment into irradiated recipients, confirming in vivo activity of Mpl-TR in HSCs. To determine whether Ott1 complexes with Mpl RNA, RNA-immunoprecipitation was performed using an HA-tagged Ott1 and revealed complex formation with Mpl RNA. Alternative splicing is frequently regulated through a co-transcriptional mechanism utilizing local epigenetic modifications including histone acetylation and H3K4me3 marks. Ott1 was previously shown to bind class I Histone deacetylases (Hdacs) and the histone H3K4 methyl-transferase (HMT), Setd1b. To establish whether Ott1 interacts with the c-Mpl gene, Chromatin-immunoprecipitation (ChIP) using HA-tagged Ott1 was performed and found binding within regions flanking the alternatively spliced exons. ChIP using anti-pan-acetyl-H4 in Ott1 knockout Lin- bone marrow showed increased histone acetylation in the region shown to bind Ott1 compared to wild type. Conversely, ChIP using anti-H3K4me3 in the Ott1 knockout showed decreased H3K4me3 at the site of Ott1 binding consistent with loss of Ott1-associated Hdac and HMT activity. To test the functional consequences on splicing, treatment of wild type cells with either a class I Hdac inhibitor or a HMT inhibitor was able to significantly increase the ratio of Mpl-TR isoform. In summary, Ott1 regulates the production of the alternatively spliced c-Mpl isoform, Mpl-TR, and consequently Thpo response in HSCs. Mpl-TR expression impairs physiologic HSC function for long and short term engraftment. Ott1 complexes with c-Mpl RNA and chromatin adjacent to the exons alternatively spliced in the Mpl-TR isoform and regulates histone acetylation and methylation marks associated with splice decision. Therefore, Ott1-mediated alternative splicing of Mpl may provide a novel mechanism via chromatin modification for modulating HSC maintenance and proliferation in response to Thpo. Furthermore, the ability to control Mpl alternative splicing through epigenetic inhibitors opens unique possibilities for pharmacologically manipulating HSC function in vitro or in vivo. Disclosures: No relevant conflicts of interest to declare.

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