In vitro and in vivo expression analysis of the human slow skeletal troponin I gene

Very small vertebrate exons are problematic for RNA splicing because of the proximity of their 3' and 5' splice sites. In this study, we investigated the recognition of a constitutive 7-nucleotide mini-exon from the troponin I gene that resides quite close to the adjacent upstream exon. The mini-exon failed to be included in spliced RNA when placed in a heterologous gene unless accompanied by the upstream exon. The requirement for the upstream exon disappeared when the mini-exon was internally expanded, suggesting that the splice sites bordering the mini-exon are compatible with those of other constitutive vertebrate exons and that the small size of the exon impaired inclusion. Mutation of the 5' splice site of the natural upstream exon did not result in either exon skipping or activation of a cryptic 5' splice site, the normal vertebrate phenotypes for such mutants. Instead, a spliced RNA accumulated that still contained the upstream intron. In vitro, the mini-exon failed to assemble into spliceosome complexes unless either internally expanded or accompanied by the upstream exon. Thus, impaired usage of the mini-exon in vivo was accompanied by impaired recognition in vitro, and recognition of the mini-exon was facilitated by the presence of the upstream exon in vivo and in vitro. Cumulatively, the atypical in vivo and in vitro properties of the troponin exons suggest a mechanism for the recognition of this mini-exon in which initial recognition of an exon-intron-exon unit is followed by subsequent recognition of the intron.

Download Full-text

In Vitro and In Vivo Expression Analysis of the Interferon-Inducible 203 Gene

Journal of Interferon & Cytokine Research ◽

10.1089/107999099314270 ◽

1999 ◽

Vol 19 (2) ◽

pp. 129-136 ◽

Cited By ~ 10

Author(s):

Giorgio Gribaudo ◽

Ludovica Riera ◽

Laura Hertel ◽

Santo Landolfo

Keyword(s):

Expression Analysis ◽

In Vivo Expression

Download Full-text

In vivo recognition of a vertebrate mini-exon as an exon-intron-exon unit

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.2677-2687.1993 ◽

1993 ◽

Vol 13 (5) ◽

pp. 2677-2687

Author(s):

D A Sterner ◽

S M Berget

Keyword(s):

Splice Site ◽

Rna Splicing ◽

Troponin I ◽

Exon Skipping ◽

Splice Sites ◽

Small Vertebrate ◽

Upstream Exon ◽

I Gene

Very small vertebrate exons are problematic for RNA splicing because of the proximity of their 3' and 5' splice sites. In this study, we investigated the recognition of a constitutive 7-nucleotide mini-exon from the troponin I gene that resides quite close to the adjacent upstream exon. The mini-exon failed to be included in spliced RNA when placed in a heterologous gene unless accompanied by the upstream exon. The requirement for the upstream exon disappeared when the mini-exon was internally expanded, suggesting that the splice sites bordering the mini-exon are compatible with those of other constitutive vertebrate exons and that the small size of the exon impaired inclusion. Mutation of the 5' splice site of the natural upstream exon did not result in either exon skipping or activation of a cryptic 5' splice site, the normal vertebrate phenotypes for such mutants. Instead, a spliced RNA accumulated that still contained the upstream intron. In vitro, the mini-exon failed to assemble into spliceosome complexes unless either internally expanded or accompanied by the upstream exon. Thus, impaired usage of the mini-exon in vivo was accompanied by impaired recognition in vitro, and recognition of the mini-exon was facilitated by the presence of the upstream exon in vivo and in vitro. Cumulatively, the atypical in vivo and in vitro properties of the troponin exons suggest a mechanism for the recognition of this mini-exon in which initial recognition of an exon-intron-exon unit is followed by subsequent recognition of the intron.

Download Full-text

In Vitro and in Vivo Calcitonin I Gene Expression in Parenchymal Cells: A Novel Product of Human Adipose Tissue

Endocrinology ◽

10.1210/en.2003-0854 ◽

2003 ◽

Vol 144 (12) ◽

pp. 5578-5584 ◽

Cited By ~ 176

Author(s):

Philippe Linscheid ◽

Dalma Seboek ◽

Eric S. Nylen ◽

Igor Langer ◽

Mirjam Schlatter ◽

...

Keyword(s):

Gene Expression ◽

Adipose Tissue ◽

Human Adipose Tissue ◽

Parenchymal Cells ◽

I Gene

Download Full-text

Inhibition of PKC phosphorylation of cTnI improves cardiac performance in vivo

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00582.2003 ◽

2004 ◽

Vol 286 (6) ◽

pp. H2089-H2095 ◽

Cited By ~ 27

Author(s):

Brian B. Roman ◽

Paul H. Goldspink ◽

Elyse Spaite ◽

Dalia Urboniene ◽

Ron McKinney ◽

...

Keyword(s):

Troponin I ◽

Cardiac Contraction ◽

Phosphorylation Sites ◽

Developed Pressure ◽

Contraction And Relaxation ◽

Intracellular Targets ◽

Camp Levels

Protein kinase C (PKC) modulates cardiomyocyte function by phosphorylation of intracellular targets including myofilament proteins. Data generated from studies on in vitro heart preparations indicate that PKC phosphorylation of troponin I (TnI), primarily via PKC-ε, may slow the rates of cardiac contraction and relaxation (+dP/d t and −dP/d t). To explore this issue in vivo, we employed transgenic mice [mutant TnI (mTnI) mice] in which the major PKC phosphorylation sites on cardiac TnI were mutated by alanine substitutions for Ser43 and Ser45 and studied in situ hemodynamics at baseline and increased inotropy. Hearts from mTnI mice exhibited increased contractility, as shown by a 30% greater +dP/dt and 18% greater −dP/d t than FVB hearts, and had a negligible response to isoproterenol compared with FVB mice, in which +dP/d t increased by 33% and −dP/d t increased by 26%. Treatment with phenylephrine and propranolol gave a similar result; FVB mouse hearts demonstrated a 20% increase in developed pressure, whereas mTnI mice showed no response. Back phosphorylation of TnI from mTnI hearts demonstrated that the mutation of the PKC sites was associated with an enhanced PKA-dependent phosphorylation independent of a change in basal cAMP levels. Our results demonstrate the important role that PKC-dependent phosphorylation of TnI has on the modulation of cardiac function under basal as well as augmented states and indicate interdependence of the phosphorylation sites of TnI in hearts beating in situ.

Download Full-text