Smaller Isoform Research Articles

Changes in protein abundance and activity involved in freezing tolerance acquisition in winter barley (Hordeum vulgare L.)

The changes in protein abundance induced by cold hardening were analysed by 2 DE in ten doubled haploid (DH) lines of winter barley, highly differentiated with respect to freezing tolerance level. Among 45 differential proteins identified by MALDI-TOF/TOF, the majority was classified as related to photosynthesis, carbohydrate metabolism, oxidation-reduction reactions and stress response. Among the detected proteins, higher abundance of RuBisCO large and small subunits, RuBisCO activase, two Oxygen-evolving enhancer proteins, Ferredoxin-NADP reductase, Cytochrome P450-dependent fatty acid hydroxylase and 14-3-3 protein was associated with higher freezing tolerance level. Lower relative level of hypothetical ATP synthase beta subunit, uncharacterized mitochondrial protein AtMg00810 and ribosomal RNA small subunit methyltransferase G also seems to be important.The results of proteomic studies were complemented by the evaluation of photosynthetic apparatus acclimation, showing distinctive differences between the studied genotypes in the number of active PSII reaction centres (RC/CSm). Additionally, the analysis of antioxidative enzyme activities suggests the importance of H2O2 as a signalling molecule possibly involved in the initiation of cold-induced plant acclimation. However, in DH lines with high freezing tolerance, H2O2 generation during cold hardening treatment was accompanied by more stable activity of catalase, H2O2-decomposing enzyme. SignificanceIn the study, the changes in protein abundance induced by cold hardening treatment were analysed by two-dimensional gel electrophoresis in ten doubled haploid (DH) lines of winter barley. Harnessing DH technology resulted in distinctive widening of genetic variation with respect to freezing tolerance level. Both the cold-hardening effect on the protein pattern in an individual winter barley DH line as well as the variation among the selected DH lines were investigated, which resulted in the identification of 45 differentiated proteins classified as involved in 14 metabolic pathways and cellular processes. Among them, eight proteins: (1) the precursor of RuBisCO large subunit, (2) RuBisCO small subunit (partial), (3) RuBisCO activase small isoform, (4) the precursor of Oxygen-evolving enhancer protein 1-like (predicted protein), (5) Oxygen-evolving enhancer protein 2, (6) the leaf isozyme of Ferredoxin-NADP reductase, (7) hypothetical protein M569_12509 Cytochrome P450-dependent fatty acid hydroxylase-like and (8) hypothetical protein BRADI_1g11290 (14-3-3 protein A-like) were accumulated to a higher level in leaves of cold-hardened seedlings of freezing tolerant winter barley DH lines in comparison with susceptible ones. Three others: (9) hypothetical protein BRADI_5g05668 F1 ATP synthase beta subunit-like, (10) predicted protein uncharacterized mitochondrial protein AtMg00810-like and (11) BnaA02g08010D Ribosomal RNA small subunit methyltransferase G-like were detected at lower level in freezing tolerant seedlings in comparison with susceptible genotypes. The last two were for the first time linked to cold acclimation. The results of complementary analyses indicate that PSII activity and stability of antioxidative enzymes under low temperature are also very important for freezing tolerance acquisition.

Novel obscurins mediate cardiomyocyte adhesion and size via the PI3K/AKT/mTOR signaling pathway

The intercalated disc of cardiac muscle embodies a highly-ordered, multifunctional network, essential for the synchronous contraction of the heart. Over 200 known proteins localize to the intercalated disc. The challenge now lies in their characterization as it relates to the coupling of neighboring cells and whole heart function. Using molecular, biochemical and imaging techniques, we characterized for the first time two small obscurin isoforms, obscurin-40 and obscurin-80, which are enriched at distinct locations of the intercalated disc. Both proteins bind specifically and directly to select phospholipids via their pleckstrin homology (PH) domain. Overexpression of either isoform or the PH-domain in cardiomyocytes results in decreased cell adhesion and size via reduced activation of the PI3K/AKT/mTOR pathway that is intimately linked to cardiac hypertrophy. In addition, obscurin-80 and obscurin-40 are significantly reduced in acute (myocardial infarction) and chronic (pressure overload) murine cardiac-stress models underscoring their key role in maintaining cardiac homeostasis. Our novel findings implicate small obscurins in the maintenance of cardiomyocyte size and coupling, and the development of heart failure by antagonizing the PI3K/AKT/mTOR pathway.

Abstract 297: GJA1-20k Protects the Heart From Ischemic Injury by Inducing Mitochondrial Metabolic Quiescence

The mRNA of the GJA1 gene that encodes gap junction protein connexin 43 (Cx43) undergoes alternative translation, producing N-terminal truncated smaller protein isoforms. Unlike the well-characterized full-length Cx43 (GJA1-43k), little is known about the function and regulation of the smaller isoforms. Here, we report that global ischemia/reperfusion injury in Langendorff-perfused mouse hearts upregulates endogenous GJA1-20k, the most abundant isoform of alternative translation. Biochemical fractionation indicates that the induced GJA1-20k is preferentially enriched in cardiomyocyte mitochondria. When introduced in vitro through adenovirus-medicated gene expression, exogenous GJA1-20k, but not full length protein GJA1-43k, localizes to mitochondria and improves the survival and viability of adult cardiomyocytes when subjected to oxidative stress. In vivo gene transfer of GJA1-20k through retro-orbital injection of AAV9 virus results in lower mitochondria-dependent basal oxygen consumption as well as maximal respiratory capacity in cardiomyocytes, unlike GFP or GJA1-43k controls. Thus GJA1-20k, but not GJA1-43k, protects the heart against ischemic injury induced by permanent LAD ligation. As compared to GFP control group, myocardial infarct size in GJA1-20k treated hearts is reduced by 30% at 72 hours post LAD ligation. These results indicate that endogenous GJA1-20k is induced upon stress and has strong tropism to mitochondria. Increased GJA1-20k induces mitochondrial metabolic quiescence and affords cardioprotection in vitro and in vivo. Alternatively translated GJA1-20k acts as a novel mitochondrial stress protein and demonstrates therapeutic potential against ischemic injury.

Abstract 235: GJA1-11k Localizes to the Nucleus and Inhibits Cell Cycle Progression

Gap Junction (GJ) channels, including the most common Connexin 43 (Cx43), are involved in the exchange of ions and small molecules to maintain homeostasis and also have fundamental roles in excitable tissues by facilitating rapid transmission of action potentials between adjacent cells. For instance, synchronization during each heartbeat is regulated by these ion channels at the cardiomyocyte cell-cell border. Numerous studies have found that Cx43 (predominantly the C-domain) is associated with control of cell proliferation, yet it is unclear how Cx43-based membrane channels effect these processes. We recently identified the existence of six endogenous Cx43 isoforms which are produced from the same full-length mRNA molecule in human cells by means of alternative translation and many of the known non-canonical roles of Cx43 can be attributed to these recently identified isoforms. Here we report that the smaller, non-channel related, alternatively translated isoform of Cx43 can directly affect cell growth. We found that 11KDa isoform (GJA1-11k) is localized in the nucleus of HEK293FT cells. GJA1-11k is a more potent growth suppressor than the wild type and full length isoform GJA1-43k. GJA1-11k-mediated growth suppression was achieved by limiting cell cycle progression from G0/G1 to S phase, in part, through regulation of internal translation of GJA1-11k. Nuclear localization experiments indicate that the cell growth-suppressive properties of GJA1-11k is linked to nuclear activity. RNA-seq based genome-wide mRNA transcriptome analysis revealed that the heat shock protein HSPA6, which is normally upregulated in certain cancer cells, was significantly downregulated in GJA1-11k overexpressing cells compared to cells overexpressing the full length isoform, suggesting a potential mechanism and/or new binding protein for the Cx43 C-terminal domain. In general, further understanding of the role of small Cx43 isoforms will provide a way not only to understand the mechanism controlling myocyte proliferation to improve the repair of the myocardium, but also to understand the pathobiology of cell growth associated with tumor suppressive properties of Cx43. It may be possible to develop new therapeutics by regulating alternative translation of Cx43.

Targeting the Role of Lipoprotein (a) in Stroke

Stroke is the third leading cause of death in the United States, behind only heart disease and cancer, with over 140,000 associated deaths per year. Considerable research is ongoing to examine the role of modifiable risk factors which may cause or contribute to stroke. Although age and family history are generally considered to be the major risk factors, there are several modifiable and non-modifiable risk factors that are linked to the pathogenesis of a stroke. Lipoprotein (a), or Lp(a), is a type of low-density lipoprotein containing an integral apolipoprotein B100 (apoB100) component with an attached apolipoprotein A-1 (ApoA-1) isoform via a disulfide linkage. Lp(a) metabolism is of great interest as it sheds light on its role in pathogenesis of not only cardiovascular disorders but also stroke. Although Lp(a) has been identified as an "LDL-like particle", its metabolism differs from low density lipoprotein (LDL). Despite some ambiguity in the literature regarding the causative effect of Lp(a) on stroke, there are clear associations of high plasma Lp(a) concentration and risk of stroke. Furthermore, the small isoforms of ApoA-1-containing lipoproteins have been shown to increase atherogenicity in atherosclerotic patients. Currently, there is little research examining the importance of small molecule Lp(a) distribution and risk for stroke, both on a first-case and recurrent basis. Understanding the role of Lp (a) in stroke requires investigating its molecular mechanisms particularly the key microRNA (s) components that control its expression and function (s). Therefore, the main objective of this review is to discuss the broader link between Lp(a) and stroke and to identify opportunities for future investigation and potential research prospects on the role of Lp (a) in stroke.

An Exportin-1–dependent microRNA biogenesis pathway during human cell quiescence

The reversible state of proliferative arrest known as "cellular quiescence" plays an important role in tissue homeostasis and stem cell biology. By analyzing the expression of miRNAs and miRNA-processing factors during quiescence in primary human fibroblasts, we identified a group of miRNAs that are induced during quiescence despite markedly reduced expression of Exportin-5, a protein required for canonical miRNA biogenesis. The biogenesis of these quiescence-induced miRNAs is independent of Exportin-5 and depends instead on Exportin-1. Moreover, these quiescence-induced primary miRNAs (pri-miRNAs) are modified with a 2,2,7-trimethylguanosine (TMG)-cap, which is known to bind Exportin-1, and knockdown of Exportin-1 or trimethylguanosine synthase 1, responsible for (TMG)-capping, inhibits their biogenesis. Surprisingly, in quiescent cells Exportin-1-dependent pri-miR-34a is present in the cytoplasm together with a small isoform of Drosha, implying the existence of a different miRNA processing pathway in these cells. Our findings suggest that during quiescence the canonical miRNA biogenesis pathway is down-regulated and specific miRNAs are generated by an alternative pathway to regulate genes involved in cellular growth arrest.

Apolipoprotein(a) inhibits hepatitis C virus entry through interaction with infectious particles.

The development of different cell culture models has greatly contributed to increased understanding of the hepatitis C virus (HCV) life cycle. However, it is still challenging to grow HCV clinical isolates in cell culture. If overcome, this would open new perspectives to study HCV biology, including drug‐resistant variants emerging with new antiviral therapies. In this study we hypothesized that this hurdle could be due to the presence of inhibitory factors in patient serum. Combining polyethylene glycol precipitation, iodixanol gradient, and size‐exclusion chromatography, we obtained from HCV‐seronegative sera a purified fraction enriched in inhibitory factors. Mass spectrometric analysis identified apolipoprotein(a) (apo[a]) as a potential inhibitor of HCV entry. Apo(a) consists of 10 kringle IV domains (KIVs), one kringle V domain, and an inactive protease domain. The 10 KIVs are present in a single copy with the exception of KIV type 2 (KIV2), which is encoded in a variable number of tandemly repeated copies, giving rise to numerous apo(a) size isoforms. In addition, apo(a) covalently links to the apolipoprotein B component of a low‐density lipoprotein through a disulfide bridge to form lipoprotein(a). Using a recombinant virus derived from the JFH1 strain, we confirmed that plasma‐derived and recombinant lipoprotein(a) as well as purified recombinant apo(a) variants were able to specifically inhibit HCV by interacting with infectious particles. Our results also suggest that small isoforms are less inhibitory than the large ones. Finally, we observed that the lipoprotein moiety of HCV lipoviroparticles was essential for inhibition, whereas functional lysine‐binding sites in KIV7, KIV8, and KIV10 were not required. Conclusions: Our results identify apo(a) as an additional component of the lipid metabolism modulating HCV infection. (Hepatology 2017;65:1851‐1864)

Novex-3, the tiny titin of muscle.

The giant multi-functional striated muscle protein titin is the third most abundant muscle protein after myosin and actin. Titin plays a pivotal role in myocardial passive stiffness, structural integrity and stress-initiated signaling pathways. The complete sequence of the human titin gene contains three isoform-specific mutually exclusive exons [termed novel exons (novex)] coding for the I-band sequence, named novex-1 (exon 45), novex-2 (exon 46) and novex-3 (exon 48). Transcripts containing either the novex-1 or novex-2 exons code for the novex-1 and novex-2 titin isoforms. The novex-3 transcript contains a stop codon and polyA tail signal, resulting in an unusually small (∼700 kDa) isoform, referred to as novex-3 titin. This 'tiny titin' isoform extends from the Z-disc (N-terminus) to novex-3 (C-terminus) and is expressed in all striated muscles. Biochemical analysis of novex-3 titin in cardiomyocytes shows that obscurin, a vertebrate muscle protein, binds to novex-3 titin. The novex-3/obscurin complex localizes to the Z-disc region and may regulate calcium, and SH3- and GTPase-associated myofibrillar signaling pathways. Therefore, novex-3 titin could be involved in stress-initiated sarcomeric restructuring.

Identification and characterization of Rubisco activase genes in Oryza punctata

AbstractRubisco activase (Rca), a specific chaperone, catalyzes the in vivo activation of Rubisco, and thus plays a major role in plant photosynthesis. Although the genes encoding Rubisco activase have been studied in many model or economic plants, few studies have analyzed their homologs in plants of closely related crop species. In this study, an Rca gene was identified and characterized in a wild relative of rice, Oryza punctata Kotschy ex Steud. The gene was 2747 bp long and possessed six exons and five introns with 47% GC content. Furthermore, cDNA sequencing produced two transcripts, RcaL and RcaS, that differed in the sequence by an inclusion of 99 bp at the carboxy terminus of RcaL. Sequence comparison between the two transcripts and the genomic DNA showed that there was a 20‐bp alternative splicing event that occurred at the fifth intron of the gene leading to the synthesis of a short polypeptide. The leaf transcriptome analysis showed that RcaS had a higher expression level than that of RcaL in a normal growth environment. In addition, the yeast two‐hybrid assays showed the small isoform of Rubisco activase in close contact with the large subunit of the Rubisco in this species, supporting the side‐on binding model of interaction between two proteins. This study broadens our understanding of the molecular characteristics of some essential genes in photosynthesis.

Cardiac Excitability at the Intercalated Disc is Maintained via a Small Isoform of Obscurin

Cardiac Excitability at the Intercalated Disc is Maintained via a Small Isoform of Obscurin

The small FOXP1 isoform predominantly expressed in activated B cell-like diffuse large B-cell lymphoma and full-length FOXP1 exert similar oncogenic and transcriptional activity in human B cells.

The forkhead transcription factor FOXP1 is generally regarded as an oncogene in activated B cell-like diffuse large B-cell lymphoma. Previous studies have suggested that a small isoform of FOXP1 rather than full-length FOXP1, may possess this oncogenic activity. Corroborating those studies, we herein show that activated B cell-like diffuse large B-cell lymphoma cell lines and primary activated B cell-like diffuse large B-cell lymphoma cells predominantly express a small FOXP1 isoform, and that the 5′-end of the Foxp1 gene is a common insertion site in murine lymphomas in leukemia virus- and transposon-mediated insertional mutagenesis screens. By combined mass spectrometry, (quantative) reverse transcription polymerase chain reaction/sequencing, and small interfering ribonucleic acid-mediated gene silencing, we determined that the small FOXP1 isoform predominantly expressed in activated B cell-like diffuse large B-cell lymphoma lacks the N-terminal 100 amino acids of full-length FOXP1. Aberrant overexpression of this FOXP1 isoform (ΔN100) in primary human B cells revealed its oncogenic capacity; it repressed apoptosis and plasma cell differentiation. However, no difference in potency was found between this small FOXP1 isoform and full-length FOXP1. Furthermore, overexpression of full-length FOXP1 or this small FOXP1 isoform in primary B cells and diffuse large B-cell lymphoma cell lines resulted in similar gene regulation. Taken together, our data indicate that this small FOXP1 isoform and full-length FOXP1 have comparable oncogenic and transcriptional activity in human B cells, suggesting that aberrant expression or overexpression of FOXP1, irrespective of the specific isoform, contributes to lymphomagenesis. These novel insights further enhance the value of FOXP1 for the diagnostics, prognostics, and treatment of diffuse large B-cell lymphoma patients.

Effects of 2,4-epibrassinolide on photosynthesis and Rubisco activase gene expression in Triticum aestivum L. seedlings under a combination of drought and heat stress

We examined the effects of 2,4-epibrassinolide (EBR) application on photosynthesis, antioxidant enzyme activity, and Rubisco activase (RCA) gene expression in wheat (Triticum aestivum L.) seedlings under a combination of drought and heat stress. The net photosynthetic rates (Pn) of wheat seedlings decreased significantly, the photosynthetic capability was inhibited, and the activities of superoxide (SOD), peroxidase (POD), catalase (CAT), and RCA as well as the initial and total activity of Rubisco declined under the combined stress. These decreases and inhibitory effects were significantly ameliorated by exogenous EBR application. Three subunits (45–46, 41–42, and 38–39 kDa) of RCA were observed in wheat seedlings. The abundances of the 38–39 kDa and 41–42 kDa subunits were significantly lower in plants subjected to stressful conditions than in unstressed plants. Interestingly, a marked increase in 45–46 kDa RCA was observed under heat or heat combined with drought stress. The abundance of 38–39 kDa RCA in seedlings exposed to heat, drought, or their combination was significantly enhanced by EBR pretreatment, which paralleled the changes in initial Rubisco activity and Pn, but was not consistent with observed mRNA abundance. These results indicated that the larger subunit of RCA (45–46 kDa), which is more thermostable and increased in response to moderate heat stress, and the smaller isoform (38–39 kDa) of RCA may play important roles in maintaining the photosynthetic capability by EBR under stress conditions.

Abstract 1269: Enhanced cytotoxicity in primary human metastatic melanoma cells via inhibition of the transient receptor potential melastatin-2 (TRPM2) channel

Abstract The transient receptor potential melastatin-2 (TRPM2) cation channel was previously identified as a potential target in various cancers, where its pharmacologic inhibition caused increased DNA damage and the selective eradication of cancer cells. In this study, we utilized several human primary metastatic melanoma cell lines, in which we analyzed TRPM2 and its isoforms, and evaluated the ability of TRPM2 inhibition to modulate cell death. As TRPM2 is known to exist as a plasma membrane cation channel in noncancerous cells, we investigated the localization of TRPM2 in melanoma cells. In three lines of primary human metastatic melanoma cells, TRPM2 was localized in the nucleus, as compared to an extra-nuclear localization in noncancerous primary epidermal keratinocyes. Because TRPM2 exists in several isoforms in cancer cells, we investigated the cellular localization of these isoforms. Subcellular fractionations demonstrated that only full-length TRPM2 was localized to the nucleus in primary human metastatic melanoma cells, whereas two smaller isoforms were localized exclusively in the cytoplasmic fraction. Treatment with the TRPM2 inhibitor, clotrimazole, caused decreased proliferation in all three lines of primary human metastatic melanoma cells. Further, treatment with the DNA alkylating agent, temozolomide (TMZ), led to increased levels of cell death in melanoma cells pretreated with clotrimazole, but not in noncancerous primary epidermal keratinocyes after pretreatment. Similar increases in cell death were observed after RNAi silencing of TRPM2 followed by TMZ treatment. Taken together, this study demonstrated that TRPM2 inhibition selectively increases cell death in primary human metastatic melanoma cells. Further, the data suggests that, similar to our previous results in breast adenocarcinoma cells, full-length TRPM2 appears to have a novel role in melanoma cell growth and survival. The results therefore suggest that TRPM2 is a potential target in melanoma, where its inhibition may cause the selective eradication of metastatic melanoma. Citation Format: David W. Koh, Steven D. Blake, Daniel P. Powell. Enhanced cytotoxicity in primary human metastatic melanoma cells via inhibition of the transient receptor potential melastatin-2 (TRPM2) channel. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1269.

Targeting of short TRPM8 isoforms induces 4TM-TRPM8-dependent apoptosis in prostate cancer cells.

Since its cloning a decade ago, TRPM8 channel has emerged as a promising prognostic marker and a putative therapeutic target in prostate cancer (PCa). However, recent studies have brought to light the complexity of TRPM8 isoforms in PCa. Consequently, the respective role of each TRPM8 isoform needs to be deciphered prior to considering TRPM8 as an attractive therapeutic target. Full-length (6 transmembrane (TM)-domain) TRPM8 channel is overexpressed in early PCa and repressed in advanced prostate tumors whereas the localization of the truncated, 4TM-TRPM8 channel (4 transmembrane (TM)-domain), in the membranes of endoplasmic reticulum (ER) is independent of the pathogenic status of epithelial cells. In the same line, expression of non-channel cytoplasmic small TRPM8 isoforms (namely sM8) is conserved in cancer cells. In this study, we identify sM8s as putative regulator of PCa cell death. Indeed, suppression of sM8 isoforms was found to induce concomitantly ER stress, oxidative stress, p21 expression and apoptosis in human epithelial prostate cancer cells. We furthermore demonstrate that induction of such mechanisms required the activity of 4TM-TRPM8 channels at the ER-mitochondria junction. Our study thus suggests that targeting sM8 could be an appropriate strategy to fight prostate cancer.

The Plastid Casein Kinase 2 Phosphorylates Rubisco Activase at the Thr-78 Site but Is Not Essential for Regulation of Rubisco Activation State.

Rubisco activase (RCA) is essential for the activation of Rubisco, the carboxylating enzyme of photosynthesis. In Arabidopsis, RCA is composed of a large RCAα and small RCAβ isoform that are formed by alternative splicing of a single gene (At2g39730). The activity of Rubisco is controlled in response to changes in irradiance by regulation of RCA activity, which is known to involve a redox-sensitive disulfide bond located in the carboxy-terminal extension of the RCAα subunit. Additionally, phosphorylation of RCA threonine-78 (Thr-78) has been reported to occur in the dark suggesting that phosphorylation may also be associated with dark-inactivation of RCA and deactivation of Rubisco. In the present study, we developed site-specific antibodies to monitor phosphorylation of RCA at the Thr-78 site and used non-reducing SDS-PAGE to monitor the redox status of the RCAα subunit. By immunoblotting, phosphorylation of both RCA isoforms occurred at low light and in the dark and feeding peroxide or DTT to leaf segments indicated that redox status of the chloroplast stroma was a critical factor controlling RCA phosphorylation. Use of a knockout mutant identified the plastid-targeted casein kinase 2 (cpCK2α) as the major protein kinase involved in RCA phosphorylation. Studies with recombinant cpCK2α and synthetic peptide substrates identified acidic residues at the –1, +2, and +3 positions surrounding Thr-78 as strong positive recognition elements. The cpck2 knockout mutant had strongly reduced phosphorylation at the Thr-78 site but was similar to wild type plants in terms of induction kinetics of photosynthesis following transfer from darkness or low light to high light, suggesting that if phosphorylation of RCA Thr-78 plays a direct role it would be redundant to redox regulation for control of Rubisco activation state under normal conditions.

Co- and post-transcriptional regulation of Rbm5 and Rbm10 in mouse cells as evidenced by tissue-specific, developmental and disease-associated variation of splice variant and protein expression levels

BackgroundExpression and function of the two RNA binding proteins and regulators of alternative splicing, RBM5 and RBM10, have largely been studied in human tissue and cell lines. The objective of the study described herein was to examine their expression in mouse tissue, in order to lay the framework for comprehensive functional studies using mouse models. MethodsAll RNA variants of Rbm5 and Rbm10 were examined in a range of normal primary mouse tissues. RNA and protein were examined in differentiating C2C12 myoblasts and in denervated and dystonin-deficient mouse skeletal muscle. ResultsAll Rbm5 and Rbm10 variants examined were expressed in all mouse tissues and cell lines. In general, Rbm5 and Rbm10 RNA expression was higher in brain than in skin. RNA expression levels were more varied between cardiac and skeletal muscle, depending on the splice variant: for instance, Rbm10v1 RNA was higher in skeletal than cardiac muscle, whereas Rbm10v3 RNA was higher in cardiac than skeletal muscle. In mouse brain, cardiac and skeletal muscle, RNA encoding an approximately 17kDa potential paralogue of a small human RBM10 isoform was detected, and the protein observed in myoblasts and myotubes. Expression of Rbm5 and Rbm10 RNA remained constant during C2C12 myogenesis, but protein levels significantly decreased. In two muscle disease models, neither Rbm10 nor Rbm5 showed significant transcriptional changes, although significant specific alternative splicing changes of Rbm5 pre-mRNA were observed. Increased RBM10 protein levels were observed following denervation. ConclusionsThe varied co-transcriptional and post-transcriptional regulation aspects of Rbm5 and Rbm10 expression associated with mouse tissues, myogenesis and muscle disease states suggest that a mouse model would be an interesting and useful model in which to study comprehensive functional aspects of RBM5 and RBM10.

A New View of Ras Isoforms in Cancers.

Does small GTPase K-Ras4A have a single state or two states, one resembling K-Ras4B and the other N-Ras? A recent study of K-Ras4A made the remarkable observation that even in the absence of the palmitoyl, K-Ras4A can be active at the plasma membrane. Importantly, this suggests that K-Ras4A may exist in two distinct signaling states. In state 1, K-Ras4A is only farnesylated, like K-Ras4B; in state 2, farnesylated and palmitoylated, like N-Ras. The K-Ras4A hypervariable region sequence is positively charged, in between K-Ras4B and N-Ras. Taken together, this raises the possibility that the farnesylated but nonpalmitoylated state 1, like K-Ras4B, binds calmodulin and is associated with colorectal and other adenocarcinomas like lung cancer and pancreatic ductal adenocarcinoma. On the other hand, state 2 may be associated with melanoma and other cancers where N-Ras is a major contributor, such as acute myeloid leukemia. Importantly, H-Ras has two, singly and doubly, palmitoylated states that may also serve distinct functional roles. The multiple signaling states of palmitoylated Ras isoforms question the completeness of small GTPase Ras isoform statistics in different cancer types and call for reevaluation of concepts and protocols. They may also call for reconsideration of oncogenic Ras therapeutics.

SKAP, an outer kinetochore protein, is required for mouse germ cell development.

In sexually reproducing organisms, accurate gametogenesis is crucial for the transmission of genetic material from one generation to the next. This requires the faithful segregation of chromosomes during mitotic and meiotic divisions. One of the main players in this process is the kinetochore, a large multi-protein complex that forms at the interface of centromeres and microtubules. Here, we analyzed the expression profile and function of small kinetochore-associated protein (SKAP) in the mouse. We found that two distinct SKAP isoforms are specifically expressed in the germline: a smaller isoform, which is detected in spermatogonia and spermatocytes and localized in the outer mitotic and meiotic kinetochores from metaphase to telophase, and a larger isoform, which is expressed in the cytoplasm of elongating spermatids. We generated SKAP-deficient mice and found that testis size and sperm production were severely reduced in mutant males. This phenotype was partially caused by defects during spermatogonia proliferation before entry into meiosis. We conclude that mouse SKAP, while being dispensable for somatic cell divisions, has an important role in the successful outcome of male gametogenesis. In germ cells, analogous to what has been suggested in studies using immortalized cells, SKAP most likely stabilizes the interaction between kinetochores and microtubules, where it might be needed as an extra safeguard to ensure the correct segregation of mitotic and meiotic chromosomes.

C/EBPβ-LAP*/LAP Expression Is Mediated by RSK/eIF4B-Dependent Signalling and Boosted by Increased Protein Stability in Models of Monocytic Differentiation.

The transcription factor C/EBPβ plays a key role in monocytic differentiation and inflammation. Its small isoform LIP is associated with proliferation at early premonocytic developmental stages and regulated via mTOR-dependent signalling. During later stages of (pre)monocytic differentiation there is a considerable increase in the large C/EBPβ isoforms LAP*/LAP which inhibit proliferation thus supporting terminal differentiation. Here, we showed in different models of monocytic differentiation that this dramatic increase in the LAP*/LAP protein and LAP/LIP ratio was accompanied by an only modest/retarded mRNA increase suggesting an important role for (post)translational mechanisms. We found that LAP*/LAP formation was induced via MEK/RSK-dependent cascades, whereas mTOR/S6K1 were not involved. Remarkably, LAP*/LAP expression was dependent on phosphorylated eIF4B, an acceleratory protein of RNA helicase eIF4A. PKR inhibition reduced the expression of eIF4B and C/EBPβ in an eIF2α-independent manner. Furthermore, under our conditions a marked stabilisation of LAP*/LAP protein occurred, accompanied by reduced chymotrypsin-like proteasome/calpain activities and increased calpastatin levels. Our study elucidates new signalling pathways inducing LAP*/LAP expression and indicates new alternative PKR functions in monocytes. The switch from mTOR- to RSK-mediated signalling to orchestrate eIF4B-dependent LAP*/LAP translation, accompanied by increased protein stability but only small mRNA changes, may be a prototypical example for the regulation of protein expression during selected processes of differentiation/proliferation.

The 133-kDa N-terminal domain enables myosin 15 to maintain mechanotransducing stereocilia and is essential for hearing.

The precise assembly of inner ear hair cell stereocilia into rows of increasing height is critical for mechanotransduction and the sense of hearing. Yet, how the lengths of actin-based stereocilia are regulated remains poorly understood. Mutations of the molecular motor myosin 15 stunt stereocilia growth and cause deafness. We found that hair cells express two isoforms of myosin 15 that differ by inclusion of an 133-kDa N-terminal domain, and that these isoforms can selectively traffic to different stereocilia rows. Using an isoform-specific knockout mouse, we show that hair cells expressing only the small isoform remarkably develop normal stereocilia bundles. However, a critical subset of stereocilia with active mechanotransducer channels subsequently retracts. The larger isoform with the 133-kDa N-terminal domain traffics to these specialized stereocilia and prevents disassembly of their actin core. Our results show that myosin 15 isoforms can navigate between functionally distinct classes of stereocilia, and are independently required to assemble and then maintain the intricate hair bundle architecture.