Site-directed biochemical analyses reveal that the switchable C-terminus of Rpc31 contributes to RNA polymerase III transcription initiation.

Rpc31 is a subunit in the TFIIE-related Rpc82/34/31 heterotrimeric subcomplex of Saccharomyces cerevisiae RNA polymerase III (pol III). Structural analyses of pol III have indicated that the N-terminal region of Rpc31 anchors on Rpc82 and further interacts with the polymerase core and stalk subcomplex. However, structural and functional information for the C-terminal region of Rpc31 is sparse. We conducted a mutational analysis on Rpc31, which uncovered a functional peptide adjacent to the highly conserved Asp-Glu-rich acidic C-terminus. This C-terminal peptide region, termed 'pre-acidic', is important for optimal cell growth, tRNA synthesis, and stable association of Rpc31 in the pre-initiation complex (PIC). Our site-directed photo-cross-linking to map protein interactions within the PIC reveal that this pre-acidic region specifically targets Rpc34 during transcription initiation, but also interacts with the DNA entry surface in free pol III. Thus, we have uncovered a switchable Rpc31 C-terminal region that functions in an initiation-specific protein interaction for pol III transcription.

Mutational and biophysical analyses reveal a TFIIIC binding region in the TFIIF-related Rpc53 subunit of RNA polymerase III.

The TFIIF-like Rpc53/Rpc37 heterodimer of RNA polymerase (pol) III is involved in various stages of transcription. The C-terminal region of Rpc53 dimerizes with Rpc37 to anchor on the lobe domain of the pol III cleft. However, structural and functional features of the Rpc53 N-terminal region had not been characterized previously. Here, we conducted site-directed alanine replacement mutagenesis on the Rpc53 N-terminus, generating yeast strains that exhibited a cold-sensitive growth defect and severely compromised pol III transcriptional activity. Circular dichroism and NMR spectroscopy revealed a highly disordered 57-amino acid polypeptide in the Rpc53 N-terminus. This polypeptide is a versatile protein-binding module displaying nanomolar-level binding affinities for Rpc37 and the Tfc4 subunit of the transcription initiation factor TFIIIC. Accordingly, we denote this Rpc53 N-terminus polypeptide as the TFIIIC-binding region or CBR. Alanine replacements in the CBR significantly reduced its binding affinity for Tfc4, highlighting its functional importance to cell growth and transcription in vitro. Our study reveals the functional basis for Rpc53's CBR in assembly of the pol III transcription initiation complex.

PRAP1 is a novel lipid-binding protein that promotes lipid absorption by facilitating MTTP-mediated lipid transport.

Microsomal triglyceride transfer protein (MTTP) is an endoplasmic reticulum resident protein that is essential for the assembly and secretion of triglyceride (TG)-rich, apoB-containing lipoproteins. Although the function and structure of mammalian MTTP have been extensively studied, how exactly MTTP transfers lipids to lipid acceptors and whether there are other biomolecules involved in MTTP-mediated lipid transport remain elusive. Here we identify a role in this process for the poorly characterized protein PRAP1. We report that PRAP1 and MTTP are partially colocalized in the endoplasmic reticulum. We observe that PRAP1 directly binds to TG and facilitates MTTP-mediated lipid transfer. A single amino acid mutation at position 85 (E85V) impairs PRAP1's ability to form a ternary complex with TG and MTTP, as well as impairs its ability to facilitate MTTP-mediated apoB-containing lipoprotein assembly and secretion, suggesting that the ternary complex formation is required for PRAP1 to facilitate MTTP-mediated lipid transport. PRAP1 is detectable in chylomicron/VLDL-rich plasma fractions, suggesting that MTTP recognizes PRAP1-bound TG as a cargo and transfers TG along with PRAP1 to lipid acceptors. Both PRAP1-deficient and E85V knock-in mutant mice fed a chow diet manifested an increase in the length of their small intestines, likely to compensate for challenges in absorbing lipid. Interestingly, both genetically modified mice gained significantly less body weight and fat mass when on high-fat diets compared with littermate controls and were prevented from hepatosteatosis. Together, this study provides evidence that PRAP1 plays an important role in MTTP-mediated lipid transport and lipid absorption.

Deriving a sub-nanomolar affinity peptide from TAP to enable smFRET analysis of RNA polymerase II complexes.

Our capability to visualize protein complexes such as RNA polymerase II (pol II) by single-molecule imaging techniques has largely been hampered by the absence of a simple bio-orthogonal approach for selective labeling with a fluorescent probe. Here, we modify the existing calmodulin-binding peptide (CBP) in the widely used Tandem Affinity Purification (TAP) tag to endow it with a high affinity for calmodulin (CaM) and use dye-CaM to conduct site-specific labeling of pol II. To demonstrate the single molecule applicability of this approach, we labeled the C-terminus of the Rpb9 subunit of pol II with donor-CaM and a site in TFIIF with an acceptor to generate a FRET (fluorescence resonance energy transfer) pair in the pol II-TFIIF complex. We then used total internal reflection fluorescence microscopy (TIRF) with alternating excitation to measure the single molecule FRET (smFRET) efficiency between these two sites in pol II-TFIIF. We found they exhibited a proximity consistent with that observed in the transcription pre-initiation complex by cryo-electron microscopy (cryo-EM). We further compared our non-covalent labeling approach with an enzyme-enabled covalent labeling method. The virtually indistinguishable results validate our smFRET approach and show that the observed proximity between the two sites represents a hallmark of the pol II-TFIIF complex. Taken together, we present a simple and versatile bio-orthogonal method derived from TAP to enable selective labeling of a protein complex. This method is suitable for analyzing dynamic relationships among proteins involved in transcription and it can be readily extended to many other biological processes.

Accelerated corneal endothelial cell loss in two patients with granulomatosis with polyangiitis following phacoemulsification.

BACKGROUND: Generally, the loss rate of human endothelial cells (HCEC) in routine cataract surgery is 8.5%. When the corneal endothelial cells density (ECD) drops, the HCEC may decompensate to keep cornea dehydration which leads to corneal edema. Granulomatosis with polyangiitis (GPA) is an uncommon autoimmune disease involving multiple organs including eyes such as conjunctivitis, scleritis, uveitis, and corneal ulcer. In this study, we report two cases of GPA whose corneal ECD decreased significantly after phacoemulsification cataract surgery. CASE PRESENTATION: In the first case of 69-year-old male with GPA, the ECD dropped 39.6% (OD) four months after phacoemulsification and 38.1% (OS) six months postoperatively respectively. At the final follow-up, the residual ECD was only 55% in the right eye in the 49th month, and 56% remained in the left eye in the 39th month. In the second case of 54-year old female, left ECD dropped 63.9% at the 4th month after surgery and 69.6% ECD remained at the 15th month postoperatively while similar ECD of right eye before and after left eye surgery. CONCLUSION: Extensive preoperative ophthalmic evaluation and meticulous postoperative inflammation control should be applied to prevent severe loss of HCEC in GPA patients.

The TFIIE-related Rpc82 subunit of RNA polymerase III interacts with the TFIIB-related transcription factor Brf1 and the polymerase cleft for transcription initiation.

Rpc82 is a TFIIE-related subunit of the eukaryotic RNA polymerase III (pol III) complex. Rpc82 contains four winged-helix (WH) domains and a C-terminal coiled-coil domain. Structural resolution of the pol III complex indicated that Rpc82 anchors on the clamp domain of the pol III cleft to interact with the duplex DNA downstream of the transcription bubble. However, whether Rpc82 interacts with a transcription factor is still not known. Here, we report that a structurally disordered insertion in the third WH domain of Rpc82 is important for cell growth and in vitro transcription activity. Site-specific photo-crosslinking analysis indicated that the WH3 insertion interacts with the TFIIB-related transcription factor Brf1 within the pre-initiation complex (PIC). Moreover, crosslinking and hydroxyl radical probing analyses revealed Rpc82 interactions with the upstream DNA and the protrusion and wall domains of the pol III cleft. Our genetic and biochemical analyses thus provide new molecular insights into the function of Rpc82 in pol III transcription.

Changes in corneal endothelial density following scleral buckling surgery for rhegmatogenous retinal detachment: a retrospective cross-sectional study.

BACKGROUND: To investigate the effect of scleral buckling (SB) on the morphology and density of human corneal endothelial cells (HCEC). METHODS: In this retrospective cross-sectional study, 26 patients who had undergone SB due to rhegmatogenous retinal detachment were enrolled, in which 15 patients received encircling while the other 11 segment types of SB. The postoperative status of affected eye, preoperative status of affected eye, and the contralateral healthy eye was served as the study, control and contralateral groups. The images of the corneal endothelium was obtained by specular microscopy at least three months postoperatively and analyzed. RESULTS: Postoperative best-corrected visual acuity of the study group was worse than that of another two groups (P < 0.001) while intraocular pressure and biometry data were similar. The mean cell area and standard deviation were larger in the study group while the coefficient of variation revealed no difference. The study group manifested a lower endothelial cell density than that of the control and the contralateral (P < 0.001) groups. Concerning the percentage of hexagonal cells, the study group showed a lower hexagonality than the control group (P = 0.04). No difference of the endothelial morphology was found between the segmental subgroup and the encircling subgroup, nor was a significant difference about endothelial cell loss found in the study group with different measurement interval. CONCLUSIONS: Scleral buckling leads to short-term decreased endothelial cell density and hexagonality, while the rest of morphological features remain unchanged. Moreover, both the segmental and encircling SB procedures yield similar postoperative HCEC status.

Ty1 Integrase Interacts with RNA Polymerase III-specific Subcomplexes to Promote Insertion of Ty1 Elements Upstream of Polymerase (Pol) III-transcribed Genes.

Retrotransposons are eukaryotic mobile genetic elements that transpose by reverse transcription of an RNA intermediate and are derived from retroviruses. The Ty1 retrotransposon of Saccharomyces cerevisiae belongs to the Ty1/Copia superfamily, which is present in every eukaryotic genome. Insertion of Ty1 elements into the S. cerevisiae genome, which occurs upstream of genes transcribed by RNA Pol III, requires the Ty1 element-encoded integrase (IN) protein. Here, we report that Ty1-IN interacts in vivo and in vitro with RNA Pol III-specific subunits to mediate insertion of Ty1 elements upstream of Pol III-transcribed genes. Purification of Ty1-IN from yeast cells followed by mass spectrometry (MS) analysis identified an enrichment of peptides corresponding to the Rpc82/34/31 and Rpc53/37 Pol III-specific subcomplexes. GFP-Trap purification of multiple GFP-tagged RNA Pol III subunits from yeast extracts revealed that the majority of Pol III subunits co-purify with Ty1-IN but not two other complexes required for Pol III transcription, transcription initiation factors (TF) IIIB and IIIC. In vitro binding studies with bacterially purified RNA Pol III proteins demonstrate that Rpc31, Rpc34, and Rpc53 interact directly with Ty1-IN. Deletion of the N-terminal 280 amino acids of Rpc53 abrogates insertion of Ty1 elements upstream of the hot spot SUF16 tRNA locus and abolishes the interaction of Ty1-IN with Rpc37. The Rpc53/37 complex therefore has an important role in targeting Ty1-IN to insert Ty1 elements upstream of Pol III-transcribed genes.

Comparative effectiveness of angiotensin-converting-enzyme inhibitors and angiotensin II receptor blockers in patients with type 2 diabetes and retinopathy.

BACKGROUND: Angiotensin-converting-enzyme (ACE) inhibitors and angiotensin II receptor blockers (ARBs) are effective treatments for diabetic retinopathy, but randomized trials and meta-analyses comparing their effects on macrovascular complications have yielded conflicting results. We compared the effectiveness of these drugs in patients with pre-existing diabetic retinopathy in a large population-based cohort. METHODS: We conducted a propensity score-matched cohort study using Taiwan's National Health Insurance Research Database. We included adult patients prescribed an ACE inhibitor or ARB within 90 days after diagnosis of diabetic retinopathy between 2000 and 2010. Primary outcomes were all-cause death and major adverse cardiovascular events (myocardial infarction, ischemic stroke or cardiovascular death). Secondary outcomes were hospital admissions with acute kidney injury or hyperkalemia. RESULTS: We identified 11 246 patients receiving ACE inhibitors and 15 173 receiving ARBs, of whom 9769 patients in each group were matched successfully by propensity scores. In the intention-to-treat analyses, ARBs were similar to ACE inhibitors in risk of all-cause death (hazard ratio [HR] 0.94, 95% confidence interval [CI] 0.87-1.01) and major adverse cardiovascular events (HR 0.95, 95% CI 0.87-1.04), including myocardial infarction (HR 1.03, 95% CI 0.88-1.20), ischemic stroke (HR 0.94, 95% CI 0.85-1.04) and cardiovascular death (HR 1.01, 95% CI 0.88-1.16). They also did not differ from ACE inhibitors in risk of hospital admission with acute kidney injury (HR 1.01, 95% CI 0.91-1.13) and hospital admission with hyperkalemia (HR 1.01, 95% CI 0.86-1.18). Results were similar in as-treated analyses. INTERPRETATION: Our study showed that ACE inhibitors were similar to ARBs in risk of all-cause death, major adverse cardiovascular events and adverse effects among patients with pre-existing diabetic retinopathy.

RNA polymerase III subunit architecture and implications for open promoter complex formation.

Transcription initiation by eukaryotic RNA polymerase (Pol) III relies on the TFIIE-related subcomplex C82/34/31. Here we combine cross-linking and hydroxyl radical probing to position the C82/34/31 subcomplex around the Pol III active center cleft. The extended winged helix (WH) domains 1 and 4 of C82 localize to the polymerase domains clamp head and clamp core, respectively, and the two WH domains of C34 span the polymerase cleft from the coiled-coil region of the clamp to the protrusion. The WH domains of C82 and C34 apparently cooperate with other mobile regions flanking the cleft during promoter DNA binding, opening, and loading. Together with published data, our results complete the subunit architecture of Pol III and indicate that all TFIIE-related components of eukaryotic and archaeal transcription systems adopt an evolutionarily conserved location in the upper part of the cleft that supports their functions in open promoter complex formation and stabilization.

Position of the general transcription factor TFIIF within the RNA polymerase II transcription preinitiation complex.

The RNA polymerase (pol) II general transcription factor TFIIF functions at several steps in transcription initiation including preinitiation complex (PIC) formation and start site selection. We find that two structured TFIIF domains bind Pol II at separate locations far from the active site with the TFIIF dimerization domain on the Pol II lobe and the winged helix domain of the TFIIF small subunit Tfg2 above the Pol II protrusion where it may interact with upstream promoter DNA. Binding of the winged helix to the protrusion is PIC specific. Anchoring of these two structured TFIIF domains at separate sites locates an essential and unstructured region of Tfg2 near the Pol II active site cleft where it may interact with flexible regions of Pol II and the general factor TFIIB to promote initiation and start site selection. Consistent with this mechanism, mutations far from the enzyme active site, which alter the binding of either structured TFIIF domains to Pol II, have similar defects in transcription start site usage.

Structural and functional characterization of interactions involving the Tfb1 subunit of TFIIH and the NER factor Rad2.

The general transcription factor IIH (TFIIH) plays crucial roles in transcription as part of the pre-initiation complex (PIC) and in DNA repair as part of the nucleotide excision repair (NER) machinery. During NER, TFIIH recruits the 3'-endonuclease Rad2 to damaged DNA. In this manuscript, we functionally and structurally characterized the interaction between the Tfb1 subunit of TFIIH and Rad2. We show that deletion of either the PH domain of Tfb1 (Tfb1PH) or several segments of the Rad2 spacer region yield yeast with enhanced sensitivity to UV irradiation. Isothermal titration calorimetry studies demonstrate that two acidic segments of the Rad2 spacer bind to Tfb1PH with nanomolar affinity. Structure determination of a Rad2-Tfb1PH complex indicates that Rad2 binds to TFIIH using a similar motif as TFIIEα uses to bind TFIIH in the PIC. Together, these results provide a mechanistic bridge between the role of TFIIH in transcription and DNA repair.

UBC9 autosumoylation negatively regulates sumoylation of septins in Saccharomyces cerevisiae.

Sumoylation regulates a wide range of cellular processes. However, little is known about the regulation of the SUMO machinery. In this study, we demonstrate that two lysine residues (Lys-153 and Lys-157) in the C-terminal region of the yeast E2-conjugating enzyme Ubc9 are the major and minor autosumoylation sites, respectively. Surprisingly, mutation of Lys-157 (ubc9(K157R)) significantly stimulates the level of Ubc9 autosumoylation at Lys-153. The functional role of Ubc9 autosumoylation is exemplified in our findings that cell cycle-dependent sumoylation of cytoskeletal septin proteins is inversely correlated with the Ubc9 autosumoylation level and that mutation of the Ubc9 autosumoylation sites results in aberrant cell morphology. Our study elucidates a regulatory mechanism that utilizes automodification of the E2 enzyme of the sumoylation machinery to control substrate sumoylation.

Intragenic transcription of a noncoding RNA modulates expression of ASP3 in budding yeast.

Inter- and intragenic noncoding transcription is widespread in eukaryotic genomes; however, the purpose of these types of transcription is still poorly understood. Here, we show that intragenic sense-oriented transcription within the budding yeast ASP3 coding region regulates a constitutively and immediately accessible promoter for the transcription of full-length ASP3. Expression of this short intragenic transcript is independent of GATA transcription factors, which are essential for the activation of full-length ASP3, and independent of RNA polymerase II (RNAPII). Furthermore, we found that an intragenic control element is required for the expression of this noncoding RNA (ncRNA). Continuous expression of the short ncRNA maintains a high level of trimethylation of histone H3 at lysine 4 (H3K4me3) at the ASP3 promoter and makes this region more accessible for RNAPII to transcribe the full-length ASP3. Our results show for the first time that intragenic noncoding transcription promotes gene expression.

The positions of TFIIF and TFIIE in the RNA polymerase II transcription preinitiation complex.

We incorporated the non-natural photoreactive amino acid p-benzoyl-L-phenylalanine (Bpa) into the RNA polymerase II (Pol II) surface surrounding the central cleft formed by the Rpb1 and Rpb2 subunits. Photo-cross-linking of preinitiation complexes (PICs) with these Pol II derivatives and hydroxyl-radical cleavage assays revealed that the TFIIF dimerization domain interacts with the Rpb2 lobe and protrusion domains adjacent to Rpb9, while TFIIE cross-links to the Rpb1 clamp domain on the opposite side of the Pol II central cleft. Mutations in the Rpb2 lobe and protrusion domains alter both Pol II-TFIIF binding and the transcription start site, a phenotype associated with mutations in TFIIF, Rpb9 and TFIIB. Together with previous biochemical and structural studies, these findings illuminate the structural organization of the PIC and the network of protein-protein interactions involved in transcription start site selection.

Heterogeneous photo-catalysis system for the degradation of azo dye Reactive Black 5 (RB5).

This study investigated a heterogeneous photo-catalysis system by introducing a novel brick supported iron oxide (denoted as B1) for the heterogeneous photoassisted degradation of Reactive Black 5 (RB5) at pH value from 3 to 7 in a three-phase (gas-liquid-solid) fluidized bed reactor (3P-FBR). Scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), X-ray powder diffraction (XRD) and N(2) adsorption/desorption were used to characterize the B1 catalyst. The in situ formation of hydrogen peroxide and the depletion of oxalic acid by photochemical cycle of Fe(III)-oxalate complex under UVA light (λ = 365 nm) were studied. The effects of the solution pH and the concentration of oxalic acid on the degradation of RB5 are elucidated. About 90% decolourization was measured and 80% of the total organic carbon (TOC) was eliminated at pH 5.0 after 120 min for 20 mg/L RB5 in presence of 10 g/L B1 catalyst, 30 mg/L oxalic acid under 15 W UVA light. A mechanism for the photocatalytic degradation of RB5 over B1 catalyst is proposed.

Sporadic zonular cataract found by scleral penetration.

PURPOSE: To present a case of bilateral zonular cataract without visual deterioration. OBSERVATION: A 41-year-old Taiwanese Han male presented with severe left ocular pain after being hit by iron filings while working. The iron dust was removed in a local hospital but due to a scleral laceration diagnosis he was referred to our hospital for further management. On examination, best-corrected visual acuity (BCVA) of 20/25 in the right eye and 20/200 in the left eye were obtained, while the before BCVA was 20/25 in the both eyes. The slit-lamp biomicroscopy demonstrated bilateral zonular cataract and left scleral full-thickness laceration with vitreous prolapse. The scleral suture procedure was then performed smoothly and the patient discharged three days later with BCVA in the left eye recovered to 20/30. CONCLUSION AND IMPORTANCE: The bilateral zonular cataract without associated systemic disorder is a rare form, which may be found incidentally with no visual symptoms.

Corneal Sensitivity and Tear Function in Recurrent Corneal Erosion Syndrome.

Purpose: To determine the association of corneal sensitivity and tear functions on the prognosis of eyes after posttraumatic recurrent corneal erosion syndrome (RCES). Methods: Patients were enrolled retrospectively and had unilateral RCES and a history of ocular surface trauma. A corneal sensitivity test and tear function test (tear break-up time and Schirmer test) were performed at three time points (month 1 to month 3, month 3 to month 6, and month 6 to month 12). Depending on the number of recurrences during the follow-up, patients were divided into group A (n > 2) or group B (n = 2). A comparison between diseased and normal fellow eyes in each patient was performed. Results: A total of 31 patients were enrolled and divided into group A (n = 14) and group B (n = 17). The mean age was 40.3 ± 12.2 years, whereas the mean follow-up was 28.0 ± 3.6 months. During the study period, corneal sensitivity, tear break-up time, and the Schirmer test results were all lower in diseased eyes than in normal fellow eyes in both groups. Compared to the first time point, recovery of corneal sensitivity and the Schirmer test values were observed in diseased eyes in group B at the second and third time points. Conclusions: Poor corneal sensitivity and tear function are associated with posttraumatic RCES. Recovery of corneal sensitivity and tear function may be associated with a reduction of recurrence in eyes with posttraumatic RCES.

Perioperative topical ascorbic acid for the prevention of phacoemulsification-related corneal endothelial damage: Two case reports and review of literature.

BACKGROUND: The current case report describes successful phacoemulsification with the aid of perioperative topical ascorbic acid (AA) in two patients with corneal endothelial disorders to prevent postoperative corneal endothelial decompensation. CASE SUMMARY: Two eyes of two patients underwent phacoemulsification with pre-existing corneal endothelial disorders including Fuchs corneal endothelial dystrophy (Patient 1) and endotheliitis (Patient 2). Topical AA was applied to both patients at least one month before and after with a frequency of four times per day. After the surgery, both eyes improved best-corrected visual acuity (BCVA) and there was limited human corneal endothelial cell loss without signs of corneal endothelial decompensation, such as deteriorated BCVA or persistent corneal edema during the follow-up of at least two years. CONCLUSION: Perioperative administration of topical AA may be an alternative therapy to the triple procedure in patients expecting to undergo cataract surgery.