Splicing Of Messenger Ribonucleic Acid Research Articles

Single Nucleotide Polymorphisms: A Deep Consideration of Protein Sequence Variation

Introduction: Human genome consists of the three billion base pairs that has about one percent of genetic variation from one person to another، which determines physical، psychological، and susceptibility to diseases. Among the types of genetic diversity, single nucleotide polymorphisms are one of the most important genetic differences between two people. Single nucleotide polymorphism variation is located in the promoter region, exons، introns، untranslated regions and other Deoxyribonucleic acid (DNA) regions. While variation in the exon region can change susceptibility to diseases depending on whether it changes the protein structure or affects translation kinetics. Diversity in the promoter region can affect the interaction of genetic and epigenetic elements. Also، variation in the promoter region can affect the DNA methylation status. Polymorphic variation in the intron region can affect Messenger Ribonucleic acid splicing and the function of cis-regulatory elements. Polymorphic variation in the 5' Untranslated region، region causes a change in translation efficiency,، while a change in the 3' Untranslated region binds micro Ribonucleic acids to their position then affects the effects. In some cases، variations in Transfer Ribonucleic acid (tRNA) and Ribosomal ribonucleic acid (rRNA) affect the function of these regulatory cis elements. Conclusion: From a clinical point of view, a deep knowledge of this type of genetic variation can help the treatment process, manage patients and understand the prognosis based on these SNPs. Private or personalized medicine is also fundamentally based on genetic diversity. In this article, it was reviewed the types of single nucleotide genetic variation and presented examples of types of cancer, neurological and immunological diseases.

Comprehensive Assessment of BARD1 Messenger Ribonucleic Acid Splicing With Implications for Variant Classification.

Introduction: Case–control analyses have shown BARD1 variants to be associated with up to >2-fold increase in risk of breast cancer, and potentially greater risk of triple negative breast cancer. BARD1 is included in several gene sequencing panels currently marketed for the prediction of risk of cancer, however there are no gene-specific guidelines for the classification of BARD1 variants. We present the most comprehensive assessment of BARD1 messenger RNA splicing, and demonstrate the application of these data for the classification of truncating and splice site variants according to American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines. Methods: Nanopore sequencing, short-read RNA-seq (whole transcriptome and targeted), and capillary electrophoresis analysis were performed by four laboratories to investigate alternative BARD1 splicing in blood, breast, and fimbriae/ovary related specimens from non-cancer affected tissues. Splicing data were also collated from published studies of nine different tissues. The impact of the findings for PVS1 annotation was assessed for truncating and splice site variants. Results: We identified 62 naturally occurring alternative spliced BARD1 splicing events, including 19 novel events found by next generation sequencing and/or reverse transcription PCR analysis performed for this study. Quantitative analysis showed that naturally occurring splicing events causing loss of clinically relevant domains or nonsense mediated decay can constitute up to 11.9% of overlapping natural junctions, suggesting that aberrant splicing can be tolerated up to this level. Nanopore sequencing of whole BARD1 transcripts characterized 16 alternative isoforms from healthy controls, revealing that the most complex transcripts combined only two alternative splicing events. Bioinformatic analysis of ClinVar submitted variants at or near BARD1 splice sites suggest that all consensus splice site variants in BARD1 should be considered likely pathogenic, with the possible exception of variants at the donor site of exon 5. Conclusions: No BARD1 candidate rescue transcripts were identified in this study, indicating that all premature translation-termination codons variants can be annotated as PVS1. Furthermore, our analysis suggests that all donor and acceptor (IVS+/−1,2) variants can be considered PVS1 or PVS1_strong, with the exception of variants targeting the exon 5 donor site, that we recommend considering as PVS1_moderate.

Truncated μ-Opioid Receptors With 6 Transmembrane Domains Are Essential for Opioid Analgesia.

Most clinical opioids act through μ-opioid receptors. They effectively relieve pain but are limited by side effects, such as constipation, respiratory depression, dependence, and addiction. Many efforts have been made toward developing potent analgesics that lack side effects. Three-iodobenzoyl-6β-naltrexamide (IBNtxA) is a novel class of opioid active against thermal, inflammatory, and neuropathic pain, without respiratory depression, physical dependence, and reward behavior. The μ-opioid receptor (OPRM1) gene undergoes extensive alternative precursor messenger ribonucleic acid splicing, generating multiple splice variants that are conserved from rodents to humans. One type of variant is the exon 11 (E11)-associated truncated variant containing 6 transmembrane domains (6TM variant). There are 5 6TM variants in the mouse OPRM1 gene, including mMOR-1G, mMOR-1M, mMOR-1N, mMOR-1K, and mMOR-1L. Gene-targeting mouse models selectively removing 6TM variants in E11 knockout (KO) mice eliminated IBNtxA analgesia without affecting morphine analgesia. Conversely, morphine analgesia is lost in an exon 1 (E1) KO mouse that lacks all 7 transmembrane (7TM) variants but retains 6TM variant expression, while IBNtxA analgesia remains intact. Elimination of both E1 and E11 in an E1/E11 double KO mice abolishes both morphine and IBNtxA analgesia. Reconstituting expression of the 6TM variant mMOR-1G in E1/E11 KO mice through lentiviral expression rescued IBNtxA but not morphine analgesia. The aim of this study was to investigate the effect of lentiviral expression of the other 6TM variants in E1/E11 KO mice on IBNtxA analgesia. Lentiviruses expressing 6TM variants were packaged in HEK293T cells, concentrated by ultracentrifugation, and intrathecally administered 3 times. Opioid analgesia was determined using a radiant-heat tail-flick assay. Expression of lentiviral 6TM variant messenger ribonucleic acids was examined by polymerase chain reaction (PCR) or quantitative PCR. All the 6TM variants restored IBNtxA analgesia in the E1/E11 KO mouse, while morphine remained inactive. Expression of lentiviral 6TM variants was confirmed by PCR or quantitative PCR. IBNtxA median effective dose values determined from cumulative dose-response studies in the rescued mice were indistinguishable from wild-type animals. IBNtxA analgesia was maintained for up to 33 weeks in the rescue mice and was readily antagonized by the opioid antagonist levallorphan. Our study demonstrated the pharmacological relevance of mouse 6TM variants in IBNtxA analgesia and established that a common functional core of the receptors corresponding to the transmembrane domains encoded by exons 2 and 3 is sufficient for activity. Thus, 6TM variants offer potential therapeutic targets for a distinct class of analgesics that are effective against broad-spectrum pain models without many side effects associated with traditional opioids.

VASCULAR ENDOTHELIAL GROWTH FACTOR RESTORES CORPOREAL SMOOTH MUSCLE FUNCTION IN VITRO

The therapeutic use of vasculogenic growth factors has been successfully demonstrated in models of organ ischemia. We determined whether vascular endothelial growth factor (VEGF) would reverse corporeal smooth muscle dysfunction in the hypercholesterolemic rabbit model of erectile dysfunction. A total of 36 New Zealand White rabbits were fed a normal (12) or 1% cholesterol (24) diet and treated after 6 weeks with 0.9 mg. VEGF or vehicle. At 6 weeks 24 rabbits received a single intracavernous dose and 12 received a single intravenous bolus of either drug. Ten days after injection corporeal smooth muscle function was analyzed after relaxation to acetylcholine and sodium nitroprusside using isometric tension studies. Corporeal sections were assessed for smooth muscle content with f-actin staining and VEGF expression by immunohistochemical study and enzyme-linked immunosorbent assay. Endothelium dependent (acetylcholine) and nitric oxide mediated (sodium nitroprusside) smooth muscle relaxation were impaired in cholesterol fed animals (p = 0.021 and 0.003, respectively). Intracavernous VEGF treatment restored sodium nitroprusside mediated relaxation to normal (p = 0.015) and intravenous VEGF restored acetylcholine and sodium nitroprusside mediated relaxation (p = 0.014 and 0.018, respectively). Decreased smooth muscle content was noted in cholesterol fed animals versus normal diet controls (p = 0.008), which was not affected by VEGF treatment (p = 0.450). Corporeal endothelial cell content was increased after intracavernous but not intravenous VEGF treatment (p = 0.001 and 0.385, respectively). VEGF expression was augmented after treatment with recombinant VEGF (p <0.001). VEGF administration variably mitigated the impairment of corporeal smooth muscle relaxation in the hypercholesterolemic rabbit model of erectile dysfunction.

Induction of vascular endothelial growth factor and hypoxia-inducible factor-1α by global ischemia in rat brain

Vascular endothelial growth factor is an angiogenic and neurotrophic peptide whose expression is transcriptionally induced in hypoxic tissues through the action of hypoxia-inducible factor-1α. To determine if this signaling pathway is activated in the ischemic brain, and might therefore participate in adaptive processes such as angiogenesis and neuroprotection, we examined the expression of vascular endothelial growth factor and hypoxia-inducible factor-1α in cerebral cortex and hippocampus following transient global cerebral ischemia in the rat. Northern analysis showed ischemia-inducible expression of multiple vascular endothelial growth factor messenger ribonucleic acid splice variants between 4 and 24 h. Western analysis and immunocytochemistry demonstrated the concerted induction of vascular endothelial growth factor and hypoxia-inducible factor-1α in the same, apparently neuronal, cells in vulnerable regions of cortex and hippocampus after 15 min of ischemia, which persisted for as long as 4 to 72 h of reperfusion. These findings demonstrate that hypoxia-sensitive vascular endothelial growth factor signaling can be induced in neurons in global cerebral ischemia in vivo, and are consistent with the hypothesis that ischemic insults trigger hypoxia-sensing and adaptive downstream molecular responses in central neurons.

Breeding Stock-Specific Variation in Peptidylglycine -Amidating Monooxygenase Messenger Ribonucleic Acid Splicing in Rat Pituitary

Breeding Stock-Specific Variation in Peptidylglycine -Amidating Monooxygenase Messenger Ribonucleic Acid Splicing in Rat Pituitary

Breeding Stock-Specific Variation in Peptidylglycineα-Amidating Monooxygenase Messenger Ribonucleic Acid Splicing in Rat Pituitary1

Peptidylglycine alpha-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the carboxyl-terminal amidation of glycine-extended peptides in a two-step reaction involving a monooxygenase and a lyase. Several forms of PAM messenger RNA result from alternative splicing of the single copy PAM gene. The presence of alternately spliced exon A between the two enzymatic domains allows endoproteolytic cleavage to occur in selected tissues, generating soluble monooxygenase and membrane lyase from integral membrane PAM. While using an exon A antiserum, we made the unexpected observation that Charles River Sprague Dawley rats expressed forms of PAM containing exon A in their pituitaries, whereas Harlan Sprague Dawley rats did not. Forms of PAM containing exon A were expressed in the atrium and hypothalamus of both types of Sprague Dawley rat, although in different proportions. PAM transmembrane domain splicing also differed between rat breeders, and full-length PAM-1 was not prevalent in the anterior pituitary of either type of rat. Despite striking differences in PAM splicing, no differences in levels of monooxygenase or lyase activity were observed in tissue or serum samples. The splicing patterns of other alternatively spliced genes, pituitary adenylate cyclase-activating polypeptide receptor type 1 and cardiac troponin T, did not vary with rat breeder. Strain-specific variations in the splicing of transcripts such as PAM must be taken into account in analyzing the resultant proteins, and knowledge of these differences should identify variations with functional significance.

20] Principles and applications of a tat-based assay for analyzing specific RNA-protein interactions in mammalian cells

Specific ribonucleic acid (RNA)–protein interactions are critical for the posttranscriptional regulation of cellular gene expression at the levels of messenger RNA (mRNA) splicing, nuclear export, translation, and stability and are key to the replication cycle of many pathogenic viruses, including human immunodeficiency virus type 1 (HIV-1), picornaviruses, and influenza viruses. Once a cis -acting RNA regulatory element has been defined, the next step is frequently the identification and subsequent characterization of the cellular or viral protein that determines the phenotype exerted by that element. In addition to the conceptually simple but often technically challenging approach of biochemical purification based on RNA affinity, an in vivo genetic approach can also be used to this problem. However, given that many of the interesting RNA–protein interactions occur in higher eukaryotes and, more specifically, in human cells, it is apparent that a simple and sensitive assay for RNA–protein binding in these same cells would be of considerable utility. The Tat-based assay for analyzing specific RNA–protein interactions relies on the unusual ability of the HIV-1 Tat protein to potently activate transcription from the viral long terminal repeat (LTR) promoter element when targeted to a promoter-proximal RNA target sequence. Specific RNA binding by a Tat-protein Y chimera can in principle be readily detected by cotransfection of an appropriate indicator construct and chimeric Tat protein expression plasmid into a relevant cell line.

Expression of messenger ribonucleic acid splice variants for vascular endothelial growth factor in the penis of adult rats and humans.

Erectile dysfunction is often associated with problems in vascular perfusion to the erectile components of the penis. In order to better understand the factors that control vascular formation and perfusion in the erectile tissues of the penis, we have begun to characterize the expression of vascular endothelial growth factor (VEGF) in penis tissues. VEGF is one of several polypeptides that have significant angiogenic activity in vitro and in vivo. Extensive characterization of the VEGF gene and its products has shown that several different mature mRNA transcripts exist, originating from alternative splicing of the basic VEGF transcript. These variant transcripts can encode peptides with different biological activities. Penile tissue was obtained from adult rats and from human patients undergoing penile prosthesis implantation. Analysis of the forms of VEGF transcripts was performed using a reverse transcription-polymerase chain reaction technique with primer pairs derived from the first and eighth exon of the VEGF gene. The expression levels of the various isoforms in the rat penis were then quantified using RNase protection assays. Four previously described splice variants of VEGF mRNA (VEGF 120, 144, 164, 188) were detected in rat and human penile tissues. In contrast to what is seen in the rat lung, where the most abundant form of VEGF mRNA is the 188 splice isoform, VEGF 164 is the most abundant transcript detected in the penis. Finally, sequence analysis of numerous VEGF cDNA clones obtained from the rat penis demonstrated the presence of a previously undescribed VEGF splice variant that could give rise to a protein of 110 amino acid residues (VEGF 110, GenBank accession no. AF080594). In summary, a number of VEGF mRNA isoforms are expressed in the rat and human penis, with the splice variant encoding a 164-amino acid protein present in greatest abundance. This study is a prelude to attempts to genetically manipulate VEGF expression in the penis as a therapy for erectile dysfunction.

, , and Mineralocorticoid Receptor Messenger Ribonucleic Acid Splice Variants: Differential Expression and Rapid Regulation in the Developing Hippocampus

, , and Mineralocorticoid Receptor Messenger Ribonucleic Acid Splice Variants: Differential Expression and Rapid Regulation in the Developing Hippocampus

Alpha, beta, and gamma mineralocorticoid receptor messenger ribonucleic acid splice variants: differential expression and rapid regulation in the developing hippocampus.

Two different types of corticoid receptor molecules bind circulating corticosterone in brain: mineralocorticoid receptors (MR) and glucocorticoid receptors. MR exhibit the highest affinity for the endogenous glucocorticoid in the rat, corticosterone. During development, low corticosterone levels influence neurogenesis, and these effects are probably MR mediated. Three MR complementary DNA clones, alpha, beta, and gamma, have been identified in the rodent. All of these MR complementary DNA clones have identical coding regions, but differ significantly at the 5'-untranslated end. Although the functional significance of these three messenger RNA (mRNA) species remains unknown, one hypothesis is that they reflect the ability of the brain to regulate the expression of MR, allowing multiple factors to differentially control transcription in a tissue- and time-specific manner. To investigate this possibility, we examined the presence of these distinct mRNA forms in the developing rat hippocampus (HC). In situ hybridization with specific alpha, beta, and gamma complementary RNA probes was performed in the HC of 3-, 5-, 7-, 12-, 14-, 28-, 35-, and 65-day-old animals. We found that there is differential expression of these forms in each of the HC subfields from infancy to adulthood. y expression appears to be associated with periods of cell birth and increased axonal sprouting. beta expression, on the other hand, may be best linked to periods of synaptogenesis, growth of commissural and associative terminal fields, and possibly active pruning. To explore the possibility that the differential gene expression may be related to corticosterone environment, adrenalectomy was performed. A rapid modulation of the MR mRNA variants (14 h) in an age- and site-specific fashion was seen. These findings suggest that the variation in expression and regulation during development of the multiple MR transcripts could reflect a complex pattern of developmental regulation that may involve a multitude of factors unique to each postnatal age and to the different neuronal populations within the hippocampal formation.

Identification of G alpha s messenger ribonucleic acid splice variants in human granulosa cells.

Granulosa cells are essential for follicular development and corpus luteum formation and their functions are regulated by gonadotrophins through G protein-coupled receptors. The dominant second messenger pathway involves the stimulation of cyclic AMP formation by G alpha s-linked receptors. In this paper we have investigated the expression of G alpha s mRNA splice variants in relation to expression of G alpha s protein isoforms in granulosa cells obtained from patients undergoing in vitro fertilization. We have carried out ribonuclease protection assays using cRNA riboprobes which are capable of detecting all G alpha s mRNA isoforms as well as quantifying total amounts of G alpha s mRNA. Granulosa cells express the message for G alpha s-Large and G alpha s-Small and the presence of two distinct protein products was confirmed by immunoblotting using the antibody RM/1. Moreover, the data show that a significant fraction of G alpha s-Large and G alpha s-Small mRNAs contain an extra CAG codon. This should generate proteins with an extra serine residue, resulting in G alpha s variants with the consensus sequence of a protein kinase C phosphorylation site. These results highlight the possible interaction between different signalling pathways in the control of cAMP production and the need to investigate the relationship between G alpha s variants and different adenylyl cyclase isozymes in patients with normal and abnormal ovarian function.

Altered insulin receptor messenger ribonucleic acid splicing in liver is associated with deterioration of glucose tolerance in the spontaneously obese and diabetic rhesus monkey: analysis of controversy between monkey and human studies

Altered insulin receptor messenger ribonucleic acid splicing in liver is associated with deterioration of glucose tolerance in the spontaneously obese and diabetic rhesus monkey: analysis of controversy between monkey and human studies

Cell-specific and regulator-induced promoter usage and messenger ribonucleic acid splicing for parathyroid hormone-related protein

Cell-specific and regulator-induced promoter usage and messenger ribonucleic acid splicing for parathyroid hormone-related protein

Cell-specific and regulator-induced promoter usage and messenger ribonucleic acid splicing for parathyroid hormone-related protein.

PTH-related protein (PTHrP) is the principle mediator of the syndrome of humoral hypercalcemia of malignancy and has potential paracrine actions on smooth muscle, epithelial cell growth, and placental calcium transport. The human PTHrP gene is complex: a combination of three promoters, one 5' alternative splicing event and alternative 3' splicing, which produces three PTHrP isoforms (139, 141, or 173 amino acids), results in multiple PTHrP messenger RNA (mRNA) species. We employed the RT-PCR technique to identify promoter usage and splicing patterns in a range of human cell lines. Cell line-specific utilization of the promoters and the 3' alternative splicing pathways was detected among bone, breast, kidney, and lung cell lines, although each cell line could potentially produce the three PTHrP isoforms. We also determined whether some of the known regulators of PTHrP differentially modulate promoter usage or splicing patterns. Dexamethasone decreased the abundance of each of the alternative mRNA species. In contrast, epidermal growth factor and transforming growth factor-beta treatment increased the abundance of each PTHrP mRNA species, with particularly marked effects on promoter 1- and promoter 2-initiated transcripts, especially those containing exon VII or VIII. Epidermal growth factor treatment was found to alter PTHrP splicing patterns in a manner consistent with increased transcription from promoters 1 and 2 and stabilization of exon VII- and IX-containing transcripts.

Human myometrial G alpha s-small (with serine) and Gs-large (with serine) messenger ribonucleic acid splice variants promote the increased expression of 46- and 54-kilodalton G alpha s protein isoforms in pregnancy and their down-regulation during labor.

In previous studies using specific G alpha s antibodies we have identified several human myometrial G alpha s protein isoforms with molecular masses of 45, 46, 47, 54, and 58 kDa, respectively. During pregnancy, levels of the 46- and 54-kDa proteins are significantly increased compared to those in nonpregnant myometrium and then decreased at the onset of labor. In this study we investigated the expression of G alpha s messenger ribonucleic acid (mRNA) splice variants, which are generated as a result of alternative splicing of a single mRNA precursor, in term pregnancy and parturition to determine whether there was any correlation with the observed changes in G alpha s protein isoforms. A myometrial G alpha s complementary DNA was synthesized using RT-PCR and cloned into pCRtmII suitable for preparation of riboprobes for use in ribonuclease protection assays. Using this technique, we identified at least three myometrial G alpha s mRNAs, including two forms of G alpha s-Large (with or without the serine at amino acid 87) and one form of G alpha s-Small (with the serine at amino acid 72). G alpha s Small (with the serine) and G alpha s-Large (with the serine) mRNAs encode for the 46- and 54-kDa G alpha s protein isoforms, respectively, and were increased in term pregnancy and then subsequently decreased after the onset of labor. Our data suggest that posttranscriptional regulation of G alpha s mRNAs may be important in the differential expression of G alpha s protein isoforms during pregnancy and labor.

Human myometrial G alpha s-small (with serine) and Gs-large (with serine) messenger ribonucleic acid splice variants promote the increased expression of 46- and 54-kilodalton G alpha s protein isoforms in pregnancy and their down-regulation during labor

Human myometrial G alpha s-small (with serine) and Gs-large (with serine) messenger ribonucleic acid splice variants promote the increased expression of 46- and 54-kilodalton G alpha s protein isoforms in pregnancy and their down-regulation during labor

Tumor-specific expression and alternate splicing of messenger ribonucleic acid encoding activin/transforming growth factor-beta receptors in human pituitary adenomas.

Activin, a member of the transforming growth factor-beta (TGF beta) cytokine family, acts as a pituitary cell mitogen via a novel family of receptor-linked serine/threonine (Ser/Thr) kinases. Pituitary tumors synthesize activin subunits, and the autocrine action of these growth factors may modulate tumor proliferation. We, therefore, investigated the expression of activin/TGF beta type I receptor messenger ribonucleic acids (mRNAs), designated ALK1 through ALK5 (ALK = activin receptor-like kinase), and type II receptor mRNAs using RT-PCR in 34 human pituitary adenomas of all phenotypes and normal pituitary tissue. ALK2 and ALK5, specific mediators of activin and TGF beta signals, respectively, were found to be expressed only in tumor and not in normal pituitary cells, and ALK2 expression was found only in tumors of a mammosomatotroph cell lineage. ALK1, ALK3, and ALK4 mRNAs were found in both normal and neoplastic pituitary cells. The alternatively spliced cytoplasmic domain of ALK4 consists of 11 kinase subdomains, that are critical for modulating receptor function and intracellular signaling. Truncated forms of the ALK4 cytoplasmic domain lacking these subdomains may attenuate activin signal transduction and affect both tumor phenotype and proliferation via the formation of inactive type I/type II complexes. Three truncated ALK4 receptor mRNAs generated by alternate splicing of the cytoplasmic Ser/Thr kinase domain were found to be tumor specific. One of these truncated receptor mRNAs, ALK4-5, is a novel splice variant that has not been previously described. Expression of the ActRII and T beta RII type II receptor mRNAs, which specifically bind activin and TGF beta, respectively, was highly prevalent among all tumor subtypes and normal pituitary tissue. However, ActRIIB, an activin-specific type II receptor that displays a 3- to 4-fold higher affinity for ligand than ActRII, was expressed in 94% of tumors, but was not prevalent in normal tissue. These data are the first to demonstrate tumor-specific expression of Ser/Thr kinase receptors mRNAs and their splice variants in human pituitary adenomas.

Tumor-specific expression and alternate splicing of messenger ribonucleic acid encoding activin/transforming growth factor-beta receptors in human pituitary adenomas

Tumor-specific expression and alternate splicing of messenger ribonucleic acid encoding activin/transforming growth factor-beta receptors in human pituitary adenomas

Relationship between GsαMessenger Ribonucleic Acid Splice Variants and the Molecular Forms of Gsa Protein in Rat Brown Adipose Tissue*

Recent work indicates that multiple molecular forms of Gs alpha can be produced by alternative splicing of a primary Gs alpha transcript. This study examined the pattern of expression of messenger RNAs (mRNAs) encoding the large (Gs alpha-L) and small (Gs alpha-S) molecular forms of Gs alpha in several rat tissues, including brown adipose tissue. In all tissues examined, Gs alpha-L mRNA was most abundant. This was most pronounced for brain tissue (95% of total), and least for brown adipose tissue (57%). The pattern of Gs alpha isoform expression of a given tissue was strongly correlated with the Gs alpha mRNA splicing pattern in that tissue. Cold exposure and surgical denervation are treatments that have been shown to, respectively, increase or decrease total Gs alpha mRNA in rat brown adipose tissue. Despite producing up to 4-fold differences in total Gs alpha mRNA, these treatments did not affect the proportions of Gs alpha mRNA splice variants in brown fat. In contrast, perinatal stimulation of brown fat was associated with changes in the splicing pattern of Gs alpha mRNA, and these changes were reflected in Gs alpha protein expression. Specifically, during the perinatal period Gs alpha-L mRNA increased significantly but Gs alpha-L protein levels did not change, whereas Gs alpha-S mRNA did not change and Gs alpha-S protein levels significantly declined. These data provide further evidence that the increased Gs alpha mRNA levels that occur in interscapular brown adipose tissue during periods of stimulation serve to maintain membrane levels of Gs alpha protein.