Annora Thoeng

Orchestrated intron retention regulates normal granulocyte differentiation.

Intron retention (IR) is widely recognized as a consequence of mis-splicing that leads to failed excision of intronic sequences from pre-messenger RNAs. Our bioinformatic analyses of transcriptomic and proteomic data of normal white blood cell differentiation reveal IR as a physiological mechanism of gene expression control. IR regulates the expression of 86 functionally related genes, including those that determine the nuclear shape that is unique to granulocytes. Retention of introns in specific genes is associated with downregulation of splicing factors and higher GC content. IR, conserved between human and mouse, led to reduced mRNA and protein levels by triggering the nonsense-mediated decay (NMD) pathway. In contrast to the prevalent view that NMD is limited to mRNAs encoding aberrant proteins, our data establish that IR coupled with NMD is a conserved mechanism in normal granulopoiesis. Physiological IR may provide an energetically favorable level of dynamic gene expression control prior to sustained gene translation.

FUS mutations in amyotrophic lateral sclerosis: clinical, pathological, neurophysiological and genetic analysis

Objective FUS gene mutations were recently identified in familial amyotrophic lateral sclerosis (ALS). The present studies sought to define the clinical, post-mortem and neurophysiological phenotypes in ALS families with FUS mutations and to determine the frequency of FUS mutations in familial and sporadic ALS. Methods FUS was screened for mutations in familial and sporadic ALS cases. Clinical, post-mortem and neurophysiological features of large families with FUS mutations are described. Results and conclusions FUS mutations were evident in 3.2% (4/124) of familial ALS, representing the second most common gene abnormality to be described in familial ALS after SOD1. No mutations were present in 247 sporadic ALS cases. The clinical presentation in 49 affected patients was consistent with a predominantly lower motor neuron disorder, supported by post-mortem findings. Upper motor neuron involvement varied, with Wallerian degeneration of corticospinal tracts present in one post-mortem case but absent in a second case from the same family. Features of cortical hyperexcitability demonstrated upper motor neuron involvement consistent with other forms of familial and sporadic ALS. One case presented with frontotemporal dementia (FTD) indicating that this may be a rare presenting feature in families with FUS mutation. Ubiquitin-positive cytoplasmic skein-like inclusions were present in lower motor neurons, but in contrast to sporadic ALS, no TDP-43 pathology was evident. Mutation-specific clinical features were identified. Patients with a R521C mutation were significantly more likely to develop disease at a younger age, and dropped-head syndrome was a frequent feature. Reduced disease penetrance was evident among most affected families.

ASCT2/SLC1A5 controls glutamine uptake and tumour growth in triple-negative basal-like breast cancer

Alanine, serine, cysteine-preferring transporter 2 (ASCT2; SLC1A5) mediates uptake of glutamine, a conditionally essential amino acid in rapidly proliferating tumour cells. Uptake of glutamine and subsequent glutaminolysis is critical for activation of the mTORC1 nutrient-sensing pathway, which regulates cell growth and protein translation in cancer cells. This is of particular interest in breast cancer, as glutamine dependence is increased in high-risk breast cancer subtypes. Pharmacological inhibitors of ASCT2-mediated transport significantly reduced glutamine uptake in human breast cancer cell lines, leading to the suppression of mTORC1 signalling, cell growth and cell cycle progression. Notably, these effects were subtype-dependent, with ASCT2 transport critical only for triple-negative (TN) basal-like breast cancer cell growth compared with minimal effects in luminal breast cancer cells. Both stable and inducible shRNA-mediated ASCT2 knockdown confirmed that inhibiting ASCT2 function was sufficient to prevent cellular proliferation and induce rapid cell death in TN basal-like breast cancer cells, but not in luminal cells. Using a bioluminescent orthotopic xenograft mouse model, ASCT2 expression was then shown to be necessary for both successful engraftment and growth of HCC1806 TN breast cancer cells in vivo. Lower tumoral expression of ASCT2 conferred a significant survival advantage in xenografted mice. These responses remained intact in primary breast cancers, where gene expression analysis showed high expression of ASCT2 and glutamine metabolism-related genes, including GLUL and GLS, in a cohort of 90 TN breast cancer patients, as well as correlations with the transcriptional regulators, MYC and ATF4. This study provides preclinical evidence for the feasibility of novel therapies exploiting ASCT2 transporter activity in breast cancer, particularly in the high-risk basal-like subgroup of TN breast cancer where there is not only high expression of ASCT2, but also a marked reliance on its activity for sustained cellular proliferation.

Loss-of-function mutations in the glutamate transporter SLC1A1 cause human dicarboxylic aminoaciduria

Solute carrier family 1, member 1 (SLC1A1; also known as EAAT3 and EAAC1) is the major epithelial transporter of glutamate and aspartate in the kidneys and intestines of rodents. Within the brain, SLC1A1 serves as the predominant neuronal glutamate transporter and buffers the synaptic release of the excitatory neurotransmitter glutamate within the interneuronal synaptic cleft. Recent studies have also revealed that polymorphisms in SLC1A1 are associated with obsessive-compulsive disorder (OCD) in early-onset patient cohorts. Here we report that SLC1A1 mutations leading to substitution of arginine to tryptophan at position 445 (R445W) and deletion of isoleucine at position 395 (I395del) cause human dicarboxylic aminoaciduria, an autosomal recessive disorder of urinary glutamate and aspartate transport that can be associated with mental retardation. These mutations of conserved residues impeded or abrogated glutamate and cysteine transport by SLC1A1 and led to near-absent surface expression in a canine kidney cell line. These findings provide evidence that SLC1A1 is the major renal transporter of glutamate and aspartate in humans and implicate SLC1A1 in the pathogenesis of some neurological disorders.

A novel TARDBP mutation in an Australian amyotrophic lateral sclerosis kindred

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that causes loss of motor neurons. A pathological hallmark of ALS is the presence of ubiquitinated TAR DNA binding protein (TDP-43) inclusions in the cytoplasm of affected cells. Rare pathogenic mutations within the gene TARDBP that encode TDP-43 were recently reported in ALS but their functional consequences are unknown. To further investigate the pathogenic role of TDP-43 in ALS, a mutation analysis of TARDBP was performed in an Australian cohort of 74 sporadic and 30 familial ALS cases. A novel familial ALS mutation in TDP-43 was identified that substitutes a highly conserved residue (G294V) and is predicted to disrupt the glycine rich domain in the C terminus, a region that plays a role in RNA binding and is required for the exon skipping activity of TDP-43.

Intron retention is regulated by altered MeCP2-mediated splicing factor recruitment

While intron retention (IR) is considered a widely conserved and distinct mechanism of gene expression control, its regulation is poorly understood. Here we show that DNA methylation directly regulates IR. We also find reduced occupancy of MeCP2 near the splice junctions of retained introns, mirroring the reduced DNA methylation at these sites. Accordingly, MeCP2 depletion in tissues and cells enhances IR. By analysing the MeCP2 interactome using mass spectrometry and RNA co-precipitation, we demonstrate that decreased MeCP2 binding near splice junctions facilitates IR via reduced recruitment of splicing factors, including Tra2b, and increased RNA polymerase II stalling. These results suggest an association between IR and a slower rate of transcription elongation, which reflects inefficient splicing factor recruitment. In summary, our results reinforce the interdependency between alternative splicing involving IR and epigenetic controls of gene expression.

RBM3 regulates temperature sensitive miR-142–5p and miR-143 (thermomiRs), which target immune genes and control fever

Fever is commonly used to diagnose disease and is consistently associated with increased mortality in critically ill patients. However, the molecular controls of elevated body temperature are poorly understood. We discovered that the expression of RNA-binding motif protein 3 (RBM3), known to respond to cold stress and to modulate microRNA (miRNA) expression, was reduced in 30 patients with fever, and in THP-1-derived macrophages maintained at a fever-like temperature (40°C). Notably, RBM3 expression is reduced during fever whether or not infection is demonstrable. Reduced RBM3 expression resulted in increased expression of RBM3-targeted temperature-sensitive miRNAs, we termed thermomiRs. ThermomiRs such as miR-142–5p and miR-143 in turn target endogenous pyrogens including IL-6, IL6ST, TLR2, PGE2 and TNF to complete a negative feedback mechanism, which may be crucial to prevent pathological hyperthermia. Using normal PBMCs that were exogenously exposed to fever-like temperature (40°C), we further demonstrate the trend by which decreased levels of RBM3 were associated with increased levels of miR-142–5p and miR-143 and vice versa over a 24 h time course. Collectively, our results indicate the existence of a negative feedback loop that regulates fever via reduced RBM3 levels and increased expression of miR-142–5p and miR-143.

LAT1 is a putative therapeutic target in endometrioid endometrial carcinoma.

l‐type amino acid transporters (LAT1‐4) are expressed in various cancer types and are involved in the uptake of essential amino acids such as leucine. Here we investigated the expression of LAT1‐4 in endometrial adenocarcinoma and evaluated the contribution of LATs to endometrial cancer cell growth. Analysis of human gene expression data showed that all four LAT family members are expressed in endometrial adenocarcinomas. LAT1 was the most highly expressed, and showed a significant increase in both serous and endometrioid subtypes compared to normal endometrium. Endometrioid patients with the highest LAT1 levels exhibited the lowest disease‐free survival. The pan‐LAT inhibitor BCH led to a significant decrease in cell growth and spheroid area in four endometrial cancer cell lines tested in vitro. Knockdown of LAT1 by shRNA inhibited cell growth in HEC1A and Ishikawa cells, as well as inhibiting spheroid area in HEC1A cells. These data show that LAT1 plays an important role in regulating the uptake of essential amino acids such as leucine into endometrial cancer cells. Increased ability of BCH compared to LAT1 shRNA at inhibiting Ishikawa spheroid area suggests that other LAT family members may also contribute to cell growth. LAT1 inhibition may offer an effective therapeutic strategy in endometrial cancer patients whose tumours exhibit high LAT1 expression.

ASCT2 regulates glutamine uptake and cell growth in endometrial carcinoma

Glutamine commonly becomes a conditionally essential amino acid in cancer. Glutamine is supplied to the cell by transporters such as ASCT2 (SLC1A5), which is frequently upregulated in multiple cancers. Here we investigated the expression of ASCT2 in endometrial carcinoma, and evaluated the contribution of ASCT2 to glutamine uptake and endometrial cancer cell growth. Analysis of human gene expression data showed that ASCT2 was significantly upregulated in both endometrioid and serous subtypes of endometrial carcinoma, compared to normal, age-matched endometrium. Furthermore, immunohistochemical staining of primary human endometrioid adenocarcinomas showed that tumours stain positive for ASCT2 in either a uniform or mosaic expression pattern, while normal adjacent glands appeared predominantly negative for ASCT2 staining. Chemical inhibition of glutamine transport by benzylserine or GPNA led to a significant decrease in endometrial cancer cell growth and spheroid cross-sectional area. ASCT2 knockdown recapitulated the decrease of cell growth and spheroid cross-sectional area in HEC1A cells, suggesting a reliance on ASCT2-mediated glutamine uptake. ASCT2 knockdown in Ishikawa cells led to lower glutamine uptake and cell growth, but did not affect spheroid area. Ishikawa cells express higher levels of the glutamine transporter SNAT1 compared to HEC1A cells, suggesting these cells may rely on both ASCT2 and SNAT1 for glutamine uptake. Since SNAT1 is also significantly upregulated in the endometrioid and serous subtypes, these data indicate that ASCT2 and SNAT1 could be used as markers of malignancy, and/or potential therapeutic targets in patients with endometrial carcinoma.

CHMP2B mutations are not a common cause of familial or sporadic amyotrophic lateral sclerosis

Approximately 10% of cases of amyotrophic lateral sclerosis (ALS) are familial with the remainder being sporadic. Known ALS genes combined (including superoxide dismutase 1 , SOD1 ) only account for about 20% of familial ALS cases, or around 2% of all ALS cases. Frontotemporal dementia (FTD) is estimated to be associated with between 3% and 22% of cases of ALS and as many as half of ALS cases appear to have frontotemporal impairment (reviewed by Neumann and colleagues1). Families with comorbid ALS and FTD are increasingly being recognised, and linkage analysis has implicated loci on chromosome 9.1 Taken together, this strongly suggests that ALS and FTD share a common pathogenic basis. Indeed, clinical and pathological overlap has been demonstrated for these disorders. Mutations in CHMP2B have been previously described in familial FTD.2 Recently, CHMP2B mutations were also described in two unrelated cases with ALS phenotypes, each with an apparent family history of ALS spectrum disorders.3 To further investigate the possible association of CHMP2B mutations with ALS, we conducted a mutation screen of cohorts with “classic” ALS, comprising 166 ALS families and 372 cases of sporadic ALS (all familial ALS were negative for the known ALS genes Superoxide dismutase …