Disruption of the developmentally-regulated Col2a1 pre-mRNA alternative splicing switch in a transgenic knock-in mouse model

The present study describes the generation of a knock-in mouse model to address the role of type II procollagen (Col2a1) alternative splicing in skeletal development and maintenance. Alternative splicing of Col2a1 precursor mRNA is a developmentally-regulated event that only occurs in chondrogenic tissue. Normally, chondroprogenitor cells synthesize predominantly exon 2-containing mRNA isoforms (type IIA and IID) while Col2a1 mRNA devoid of exon 2 (type IIB) is the major isoform produced by differentiated chondrocytes. Another isoform, IIC, has also been identified that contains a truncated exon 2 and is not translated into protein. The biological significance of this IIA/IID to IIB splicing switch is not known. Utilizing a splice site targeting knock-in approach, a 4 nucleotide mutation was created to convert the 5' splice site of Col2a1 exon 2 from a weak, non-consensus sequence to a strong, consensus splice site. This resulted in apparent expression of only the IIA mRNA isoform, as confirmed in vitro by splicing of a type II procollagen mini-gene containing the 5' splice site mutation. To test the splice site targeting approach in vivo, homozygote mice engineered to retain IIA exon 2 (Col2a1(+ex2)) were generated. Chondrocytes from hindlimb epiphyseal cartilage of homozygote mice were shown to express only IIA mRNA and protein at all pre- and post-natal developmental stages analyzed (E12.5, E16.5, P0, P3, P7, P14, P28 and P70). As expected, type IIB procollagen was the major isoform produced in wild type cartilage at all post-natal time points. Col2a1(+ex2) homozygote mice are viable, appear healthy and display no overt phenotype to date. However, research is currently underway to investigate the biological consequence of persistent expression of the exon 2-encoded conserved cysteine-rich domain in post-natal skeletal tissues.     

Functionally diverse complement of large conductance calcium- and voltage-activated potassium channel (BK) alpha-subunits generated from a single site of splicing

The pore-forming alpha-subunits of large conductance calcium- and voltage-activated potassium (BK) channels are encoded by a single gene that undergoes extensive alternative pre-mRNA splicing. However, the extent to which differential exon usage at a single site of splicing may confer functionally distinct properties on BK channels is largely unknown. Here we demonstrated that alternative splicing at site of splicing C2 in the mouse BK channel C terminus generates five distinct splice variants: ZERO, e20, e21(STREX), e22, and a novel variant deltae23. Splice variants display distinct patterns of tissue distribution with e21(STREX) expressed at the highest levels in adult endocrine tissues and e22 at embryonic stages of mouse development. deltae23 is not functionally expressed at the cell surface and acts as a dominant negative of cell surface expression by trapping other BK channel splice variant alpha-subunits in the endoplasmic reticulum and perinuclear compartments. Splice variants display a range of biophysical properties. e21(STREX) and e22 variants display a significant left shift (>20 mV at 1 microM [Ca2+]i) in half-maximal voltage of activation compared with ZERO and e20 as well as considerably slower rates of deactivation. Splice variants are differentially sensitive to phosphorylation by endogenous cAMP-dependent protein kinase; ZERO, e20, and e22 variants are all activated, whereas e21 (STREX) is the only variant that is inhibited. Thus alternative pre-mRNA splicing from a single site of splicing provides a mechanism to generate a physiologically diverse complement of BK channel alpha-subunits that differ dramatically in their tissue distribution, trafficking, and regulation.     

Alternative splicing attenuates transgenic expression directed by the apolipoprotein E promoter-enhancer based expression vector pLIV11

The plasmid vector pLIV11 is used commonly to achieve liver-specific expression of genes of interest in transgenic mice and rabbits. Expression is driven by the human apolipoprotein (apo)E 5′ proximal promoter, which includes 5 kb of upstream sequence, exon 1, intron 1, and 5 bp of exon 2. A 3.8 kb 3′ hepatic control region, derived from a region ∼18 kb downstream of the apoE gene, enhances liver-specific expression. Here, we report that cDNA sequences inserted into the multiple cloning site (MCS) of pLIV11, which is positioned just downstream of truncated exon 2, can cause exon 2 skipping. Hence, splicing is displaced to downstream cryptic 3′ splice acceptor sites causing deletion of cloned 5′ untranslated mRNA sequences and, in some cases, deletion of the 5′ end of an open reading frame. To prevent use of cryptic splice sites, the pLIV11 vector was modified with an engineered 3′ splice acceptor site inserted immediately downstream of truncated apoE exon 2. Presence of this sequence fully shifted splicing of exon 1 from the native intron 1–exon 2 splice acceptor site to the engineered site. This finding confirmed that sequences inserted into the MCS of the vector pLIV11 can affect exon 2 recognition and provides a strategy to protect cloned sequences from alternative splicing and possible attenuation of transgenic expression.     

Alternative splicing of type II procollagen exon 2 is regulated by the combination of a weak 5' splice site and an adjacent intronic stem-loop cis element

Alternative splicing of the type II procollagen gene (COL2A1) is developmentally regulated during chondrogenesis. Chondroprogenitor cells produce the type IIA procollagen isoform by splicing (including) exon 2 during pre-mRNA processing, whereas differentiated chondrocytes synthesize the type IIB procollagen isoform by exon 2 skipping (exclusion). Using a COL2A1 mini-gene and chondrocytes at various stages of differentiation, we identified a non-classical consensus splicing sequence in intron 2 adjacent to the 5' splice site, which is essential in regulating exon 2 splicing. RNA mapping confirmed this region contains secondary structure in the form of a stem-loop. Mutational analysis identified three cis elements within the conserved double-stranded stem region that are functional only in the context of the natural weak 5' splice site of exon 2; they are 1) a uridine-rich enhancer element in all cell types tested except differentiated chondrocytes; 2) an adenine-rich silencer element, and 3) an enhancer cis element functional in the context of secondary structure. This is the first report identifying key cis elements in the COL2A1 gene that modulate the cell type-specific alternative splicing switch of exon 2 during cartilage development.     

Alternative splicing and gene structure of the transforming growth factor beta-activated kinase 1

We have identified a fourth splice variant of the TGF beta-activated kinase (TAK1), called TAK1-d, and identified an error in the previously published TAK1-c sequence. Our data shows that the c and d variants encode proteins whose carboxyl ends differ markedly from those of variants a and b. Analysis of the human TAK1 gene sequence, located at 6q16.1-q16.3, shows that the coding sequence is organised in 17 exons. The four splice variants result from alternative splicing of exons 12 and 16, the reading frame of exon 17 being determined by the presence or absence of exon 16. Study of the relative levels of expression of the four splice variants showed significant variations between tissues. Our evidence suggests that the alternative splicing of the TAK1 mRNA may have important functional implications.     

Insulin promotes neuronal survival via the alternatively spliced protein kinase CδII isoform

Insulin signaling pathways in the brain regulate food uptake and memory and learning. Insulin and protein kinase C (PKC) pathways are integrated and function closely together. PKC activation in the brain is essential for learning and neuronal repair. Intranasal delivery of insulin to the central nervous system (CNS) has been shown to improve memory, reduce cerebral atrophy, and reverse neurodegeneration. However, the neuronal molecular mechanisms of these effects have not been studied in depth. PKCδ plays a central role in cell survival. Its splice variants, PKCδI and PKCδII, are switches that determine cell survival and fate. PKCδI promotes apoptosis, whereas PKCδII promotes survival. Here, we demonstrate that insulin promotes alternative splicing of PKCδII isoform in HT22 cells. The expression of PKCδI splice variant remains unchanged. Insulin increases PKCδII alternative splicing via the PI3K pathway. We further demonstrate that Akt kinase mediates phosphorylation of the splicing factor SC35 to promote PKCδII alternative splicing. Using overexpression and knockdown assays, we demonstrate that insulin increases expression of Bcl2 and bcl-xL via PKCδII. We demonstrate increased cell proliferation and increased BrdU incorporation in insulin-treated cells as well as in HT22 cells overexpressing PKCδII. Finally, we demonstrate in vivo that intranasal insulin promotes cognitive function in mice with concomitant increases in PKCδII expression in the hippocampus. This is the first report of insulin, generally considered a growth or metabolic hormone, regulating the alternative isoform expression of a key signaling kinase in neuronal cells such that it results in increased neuronal survival.     

Alternative splicing in intracellular loop connecting domains II and III of the alpha 1 subunit of Cav1.2 Ca2+ channels predicts two-domain polypeptides with unique C-terminal tails

Novel splice variants of the alpha(1) subunit of the Ca(v)1.2 voltage-gated Ca(2+) channel were identified that predicted two truncated forms of the alpha(1) subunit comprising domains I and II generated by alternative splicing in the intracellular loop region linking domains II and III. In rabbit heart splice variant 1 (RH-1), exon 19 was deleted, which resulted in a reading frameshift of exon 20 with a premature termination codon and a novel 19-amino acid carboxyl-terminal tail. In the RH-2 variant, exons 17 and 18 were deleted, leading to a reading frameshift of exons 19 and 20 with a premature stop codon and a novel 62-amino acid carboxyl-terminal tail. RNase protection assays with RH-1 and RH-2 cRNA probes confirmed the expression in cardiac and neuronal tissue but not skeletal muscle. The deduced amino acid sequence from full-length cDNAs encoding the two variants predicted polypeptides of 99.0 and 99.2 kDa, which constituted domains I and II of the alpha(1) subunit of the Ca(v)1.2 channel. Antipeptide antibodies directed to sequences in the second intracellular loop between domains II and III identified the 240-kDa Ca(v)1.2 subunit in sarcolemmal and heavy sarcoplasmic reticulum (HSR) membranes and a 99-kDa polypeptide in the HSR. An antipeptide antibody raised against unique sequences in the RH-2 variant also identified a 99-kDa polypeptide in the HSR. These data reveal the expression of additional Ca(2+) channel structural units generated by alternative splicing of the Ca(v)1.2 gene.     

Inhibition of cGMP-dependent protein kinase II by its own splice isoform

cGMP- and cAMP-dependent protein kinases (cGK I, cGK II, and cAK) are important mediators of many signaling pathways that increase cyclic nucleotide concentrations and ultimately phosphorylation of substrates vital to cellular functions. Here we demonstrate a novel mRNA splice isoform of cGK II arising from alternative 5' splicing within exon 11. The novel splice variant encodes a protein (cGK II Delta(441-469)) lacking 29 amino acids of the cGK II Mg-ATP-binding/catalytic domain, including the conserved glycine-rich loop consensus motif Gly-x-Gly-x-x-Gly-x-Val which interacts with ATP in the protein kinase family of enzymes. cGK II Delta(441-469) has no intrinsic enzymatic activity itself, however, it antagonizes cGK II and cGK I, but not cAK. Thus, the activation and cellular functions of cGK II may be determined not only by intracellular cGMP levels but also by alternative splicing which may regulate the balance of expression of cGK II versus its own inhibitor, cGK II Delta(441-469).     

Functional comparison of transactivation by simian immunodeficiency virus from rhesus macaques and human immunodeficiency virus type 1.

Simian immunodeficiency virus from rhesus macaques (SIVmac), like human immunodeficiency virus type 1 (HIV-1), encodes a transactivator (tat) which stimulates long terminal repeat (LTR)-directed gene expression. We performed cotransfection assays of SIVmac and HIV-1 tat constructs with LTR-CAT reporter plasmids. The primary effect of transactivation for both SIVmac and HIV-1 is an increase in LTR-directed mRNA accumulation. The SIVmac tat gene product partially transactivates an HIV-1 LTR, whereas the HIV-1 tat gene product fully transactivates an SIVmac LTR. Significant transactivation is achieved by the product of coding exon 1 of the HIV-1 tat gene; however, inclusion of coding exon 2 results in a further increase in mRNA accumulation. In contrast, coding exon 2 of the SIVmac tat gene is required for significant transactivation. These results imply that the tat proteins of SIVmac and HIV-1 are functionally similar but not interchangeable. In addition, an in vitro-generated mutation in SIVmac tat disrupts splicing at the normal splice acceptor site at the beginning of coding exon 2 and activates a site approximately 15 nucleotides downstream. The product of this splice variant stimulates LTR-directed gene expression. This alternative splice acceptor site is also used by a biologically active provirus with an efficiency of approximately 5% compared with the upstream site. These data suggest that a novel tat protein is encoded during the course of viral infection.