Pull-down of Biotinylated RNA and Associated Proteins.

Mapping networks of RNA-protein interactions in cells is essential for understanding the inner workings of many biological processes, including RNA processing, trafficking, and translation. Current in vivo methods for studying protein-RNA interactions rely mostly on purification of poly(A) transcripts, which represent only ~2-3% of total RNAs (Figure 1). Alternate robust methods for tagging RNA molecules with an RNA aptamer (e.g., MS2-, U1A- and biotin-RNA aptamer) and capturing the RNA-protein complex by the respective aptamer-specific partner are not extensively studied. Here, we describe a protocol (Figure 2) in which a biotin-RNA aptamer, referred to as the RNA mimic of biotin (RMB), was conjugated separately to two small RNA secondary structures that contribute to trafficking and translating HAC1 mRNA in the budding yeast Saccharomyces cerevisiae. The RMB-tagged RNA was expressed in yeast cells from a constitutive promoter. The biotinylated RNA bound to proteins was pulled down from the cell lysate by streptavidin agarose beads. RNA was detected by RT-PCR (Figure 3) and associated proteins by mass spectrometry (Figure 4). Our findings show that an RNA aptamer tag to RNA molecule is an effective method to explore the functional roles of RNA-protein networks in vivo.

RNA-binding protein FXR1 drives cMYC translation by recruiting eIF4F complex to the translation start site.

Fragile X-related protein-1 (FXR1) gene is highly amplified in patients with ovarian cancer, and this amplification is associated with increased expression of both FXR1 mRNA and protein. FXR1 expression directly associates with the survival and proliferation of cancer cells. Surface sensing of translation (SUnSET) assay demonstrates that FXR1 enhances the overall translation in cancer cells. Reverse-phase protein array (RPPA) reveals that cMYC is the key target of FXR1. Mechanistically, FXR1 binds to the AU-rich elements (ARE) present within the 3' untranslated region (3'UTR) of cMYC and stabilizes its expression. In addition, the RGG domain in FXR1 interacts with eIF4A1 and eIF4E proteins. These two interactions of FXR1 result in the circularization of cMYC mRNA and facilitate the recruitment of eukaryotic translation initiation factors to the translation start site. In brief, we uncover a mechanism by which FXR1 promotes cMYC levels in cancer cells.

Phosphorylation of Pal2 by the protein kinases Kin1 and Kin2 modulates HAC1 mRNA splicing in the unfolded protein response in yeast.

During cellular stress in the budding yeast Saccharomyces cerevisiae, an endoplasmic reticulum (ER)-resident dual kinase and RNase Ire1 splices an intron from HAC1 mRNA in the cytosol, thereby releasing its translational block. Hac1 protein then activates an adaptive cellular stress response called the unfolded protein response (UPR) that maintains ER homeostasis. The polarity-inducing protein kinases Kin1 and Kin2 contribute to HAC1 mRNA processing. Here, we showed that an RNA-protein complex that included the endocytic proteins Pal1 and Pal2 mediated HAC1 mRNA splicing downstream of Kin1 and Kin2. We found that Pal1 and Pal2 bound to the 3' untranslated region (3'UTR) of HAC1 mRNA, and a yeast strain lacking both Pal1 and Pal2 was deficient in HAC1 mRNA processing. We also showed that Kin1 and Kin2 directly phosphorylated Pal2, and that a nonphosphorylatable Pal2 mutant could not rescue the UPR defect in a pal1Δ pal2Δ strain. Thus, our work uncovers a Kin1/2-Pal2 signaling pathway that coordinates HAC1 mRNA processing and ER homeostasis.

Legal support to gain access to medical treatment in the current healthcare system is unproductive.

The National Health Service in UK is facing grave financial crisis. Recently, 65% of Acute Trusts have reported a collective deficit of £2.5 billion. This financial crunch has had significant impact on patient care and sustenance of essential healthcare services. In order to thrive, the National Health Service has begun significant rationing of treatment which has become increasingly apparent in recent times, exposing the National Health Service to legal challenges. This article reviews the current state of the National Health Service from a legal perspective.

Adaptation to Endoplasmic Reticulum Stress Requires Transphosphorylation within the Activation Loop of Protein Kinases Kin1 and Kin2, Orthologs of Human Microtubule Affinity-Regulating Kinase.

Perturbations in endoplasmic reticulum (ER) homeostasis, a condition termed ER stress, activate the unfolded protein response (UPR), an intracellular network of signaling pathways. Recently, we have shown that protein kinase Kin1 and its paralog, Kin2, in the budding yeast Saccharomyces cerevisiae (orthologs of microtubule affinity-regulating kinase in humans) contribute to the UPR function. These Kin kinases contain a conserved kinase domain and an autoinhibitory kinase-associated 1 (KA1) domain separated by a long undefined domain. Here, we show that Kin1 or Kin2 protein requires minimally a kinase domain and an adjacent kinase extension region (KER) for UPR function. We also show that the functional mini-Kin2 protein is predominantly visualized inside the cells and precipitated with the cellular membrane fraction, suggesting its association with the cellular endomembrane system. Furthermore, we show that transphosphorylation of the Kin1 residue T302 and the analogous Kin2 residue T281 within the activation loop are important for full kinase activity. Collectively, our data suggest that, during ER stress, the Kin kinase domain is released from its autoinhibitory KA1 domain and is activated by transphosphorylation.

Phosphorylation of translation initiation factor eIF2α at Ser51 depends on site- and context-specific information.

Protein kinases phosphorylate specific amino acid residues of substrate proteins and regulate many cellular processes. Specificity for phosphorylation depends on the accessibility of these residues, and more importantly, kinases have preferences for certain residues flanking the phospho-acceptor site. Translation initiation factor 2α [eukaryotic translation initiation factor 2α (eIF2α)] kinase phosphorylates serine51 (Ser51) of eIF2α and downregulates cellular protein synthesis. Structural information on eIF2α reveals that Ser51 is located within a flexible loop, referred to as the Ser51 loop. Recently, we have shown that conformational change of the Ser51 loop increases the accessibility of Ser51 to the kinase active site for phosphorylation. Here, we show that the specificity of Ser51 phosphorylation depends largely on its relative position in the Ser51 loop and minimally on the flanking residues.

Financial implications of laparoscopic hot gallbladder service in a nontertiary District General Hospital.

INTRODUCTION: The service of providing index admission laparoscopic cholecystectomy (IALC), as recommended by NIC guidelines, often falls short in nontertiary centres because of a combination of limited resources and financial constraints. METHODS: This retrospective study in a single-centre District General Hospital included 50 patients, eligible to undergo IALC, and calculated potential savings from performing IALC on the day of admission by considering admission tariffs, bed, and operating costs. RESULTS: The IALC was provided in 19 patients (38%), with a mean delay from admission to operation of (median) 3 days. Mean surplus tariff was £1421 and £1571 in IALC and non-IALC groups, respectively. Performing immediate IALC (on the day of admission) for acute cholecystitis (AC) is predicted to increase mean surplus tariff to £2132 per patient, raising total predicted annual surplus by £53 000. Immediate IALC is also predicted to reduce waiting time for day-case LC by freeing up 53 day-case slots, attracting additional £95 600 annually, along with freeing up many inpatient bed days. CONCLUSIONS: This study demonstrates that reduction of preoperative stay in AC by expediting operations in every eligible patient promises significant surplus revenue. Additional advantages include reducing inpatient bed days and freeing up operating lists that are otherwise taken up by patients for interval cholecystectomy.