Identification of a quality-control mechanism for mRNA 5'-end capping.

The 7-methylguanosine cap structure at the 5' end of eukaryotic messenger RNAs is a critical determinant of their stability and translational efficiency. It is generally believed that 5'-end capping is a constitutive process that occurs during mRNA maturation and lacks the need for a quality-control mechanism to ensure its fidelity. We recently reported that the yeast Rai1 protein has pyrophosphohydrolase activity towards mRNAs lacking a 5'-end cap. Here we show that, in vitro as well as in yeast cells, Rai1 possesses a novel decapping endonuclease activity that can also remove the entire cap structure dinucleotide from an mRNA. This activity is targeted preferentially towards mRNAs with unmethylated caps in contrast to the canonical decapping enzyme, Dcp2, which targets mRNAs with a methylated cap. Capped but unmethylated mRNAs generated in yeast cells with a defect in the methyltransferase gene are more stable in a rai1-gene-disrupted background. Moreover, rai1Δ yeast cells with wild-type capping enzymes show significant accumulation of mRNAs with 5'-end capping defects under nutritional stress conditions of glucose starvation or amino acid starvation. These findings provide evidence that 5'-end capping is not a constitutive process that necessarily always proceeds to completion and demonstrates that Rai1 has an essential role in clearing mRNAs with aberrant 5'-end caps. We propose that Rai1 is involved in an as yet uncharacterized quality control process that ensures mRNA 5'-end integrity by an aberrant-cap-mediated mRNA decay mechanism.

Pathologic Findings and Management of Renal Mass in Horseshoe Kidneys.

OBJECTIVES: To evaluate the management, surgical outcomes, and pathological findings in patients with tumor in a horseshoe-kidney (HK). HK patients present unique challenges due to aberrant vascular anatomy and risk of renal insufficiency. We hypothesized that many tumors in this setting may be indolent or benign. MATERIALS AND METHODS: Patients managed for renal mass in HK at our center (1999-2021) were reviewed. Baseline characteristics, surgical approach, complications, functional outcomes, pathology, and survival were analyzed. RESULTS: Forty-three procedures were performed in 42 patients with HK including 24 nephron-sparing surgeries (NSS) and 19 radical nephrectomies (RN: splitting the isthmus and saving the contralateral moiety). NSS included 22 partial nephrectomy (PN) and 2 thermal ablations. Median tumor size was 4.3 cm. Eighteen cases (42%) were minimally-invasive, 17 open-midline, and 8 other open approaches. Ninety-day Clavien III-V complication rate was 12% with no mortalities. For PN, median warm/cold ischemia times were 26/31 minutes, respectively. On pathology, only 27 tumors (63%) were renal-cell-carcinoma (RCC), and 22 tumors (51%) were either benign (n = 10) or low grade, confined RCC (n = 12). Preoperative/new baseline/long-term eGFR were 82/83/78 mL/min/1.73 m2 after NSS vs 75/48/57 mL/min/1.73 m2 after RN, respectively. Long-term dialysis was required in 3 patients (7%). Median follow-up was 36 months. Five-year recurrence-free survival was 83% for NSS and 66% for RN. CONCLUSIONS: Management of renal masses in HK is challenging and requires versatility with multiple surgical approaches. Preservation of renal function was accomplished in most patients, with a functional advantage observed for NSS. RCC was less common than expected while benign and non-aggressive tumors were prevalent, suggesting consideration for preoperative renal-mass-biopsy when feasible.

Assessing the Status of Mentorship Programs in Interventional Radiology Residency Training: Results of a 2018 Survey.

RATIONALE AND OBJECTIVES: To assess the prevalence and structure of mentorship programs in interventional radiology (IR) residency programs. MATERIALS AND METHODS: A 12-question anonymous survey was distributed via email to all 78 program directors (PDs) of United States IR residency programs. The survey included information about the presence or absence of a formal mentorship program at their institution, how the program functions, potential barriers to implementation, and future plans for mentorship. RESULTS: Twenty-three of 78 integrated IR residency PDs completed the survey (response rate 29.5%). Thirteen of 23 reports that they currently have a formal mentorship program in place and 11 of 13 report no direct departmental support for mentorship. Of those that do not have a mentorship program in place, 5 of 10 report that implementation is underway. These programs report that the absence of a mentorship program is due to a lack of dedicated time and financial support. While 8 of 23 PDs were unaware of the Society of Interventional Radiology Mentor Match program, 6of 23 were registered as mentors through it. Nearly all PDs reported interest in receiving mentoring resources from SIR with the most popular choices being a dedicated mentorship educational course at the SIR annual meeting and regular mentorship articles and practical tips in publications such as IR quarterly. CONCLUSIONS: Despite involvement of many IR PDs in mentorship, numerous residency programs lack a formal mentorship program. Of those with a program, most don't receive direct departmental support and those without a program cite lack of time and financial support as barriers to effective implementation.

Depletion of phosphatidylcholine in yeast induces shortening and increased saturation of the lipid acyl chains: evidence for regulation of intrinsic membrane curvature in a eukaryote.

To study the consequences of depleting the major membrane phospholipid phosphatidylcholine (PC), exponentially growing cells of a yeast cho2opi3 double deletion mutant were transferred from medium containing choline to choline-free medium. Cell growth did not cease until the PC level had dropped below 2% of total phospholipids after four to five generations. Increasing contents of phosphatidylethanolamine (PE) and phosphatidylinositol made up for the loss of PC. During PC depletion, the remaining PC was subject to acyl chain remodeling with monounsaturated species replacing diunsaturated species, as shown by mass spectrometry. The remodeling of PC did not require turnover by the SPO14-encoded phospholipase D. The changes in the PC species profile were found to reflect an overall shift in the cellular acyl chain composition that exhibited a 40% increase in the ratio of C16 over C18 acyl chains, and a 10% increase in the degree of saturation. The shift was stronger in the phospholipid than in the neutral lipid fraction and strongest in the species profile of PE. The shortening and increased saturation of the PE acyl chains were shown to decrease the nonbilayer propensity of PE. The results point to a regulatory mechanism in yeast that maintains intrinsic membrane curvature in an optimal range.

Regulation of long chain unsaturated fatty acid synthesis in yeast.

Saccharomyces cerevisiae forms monounsaturated fatty acids using the ER membrane-bound Delta-9 fatty acid desaturase, Ole1p, an enzyme system that forms a double bond in saturated fatty acyl CoA substrates. Ole1p is a chimeric protein consisting of an amino terminal desaturase domain fused to cytochrome b5. It catalyzes the formation of the double bond through an oxygen-dependent mechanism that requires reducing equivalents from NADH. These are transferred to the enzyme via NADH cytochrome b5 reductase to the Ole1p cytochrome b5 domain and then to the diiron-oxo catalytic center of the enzyme. The control of OLE1 gene expression appears to mediated through the ER membrane proteins Spt23p and Mga2p. N-terminal fragments of these proteins are released by an ubiquitin/proteasome mediated proteolysis system and translocated to the nucleus where they appear to act as transcription coactivators of OLE1. OLE1 is regulated through Spt23p and Mga2p by multiple systems that control its transcription and mRNA stability in response to diverse stimuli that include nutrient fatty acids, carbon source, metal ions and the availability of oxygen.

Fusing Swarm Intelligence and Self-Assembly for Optimizing Echo State Networks.

Optimizing a neural network's topology is a difficult problem for at least two reasons: the topology space is discrete, and the quality of any given topology must be assessed by assigning many different sets of weights to its connections. These two characteristics tend to cause very "rough." objective functions. Here we demonstrate how self-assembly (SA) and particle swarm optimization (PSO) can be integrated to provide a novel and effective means of concurrently optimizing a neural network's weights and topology. Combining SA and PSO addresses two key challenges. First, it creates a more integrated representation of neural network weights and topology so that we have just a single, continuous search domain that permits "smoother" objective functions. Second, it extends the traditional focus of self-assembly, from the growth of predefined target structures, to functional self-assembly, in which growth is driven by optimality criteria defined in terms of the performance of emerging structures on predefined computational problems. Our model incorporates a new way of viewing PSO that involves a population of growing, interacting networks, as opposed to particles. The effectiveness of our method for optimizing echo state network weights and topologies is demonstrated through its performance on a number of challenging benchmark problems.

Dxo1 is a new type of eukaryotic enzyme with both decapping and 5'-3' exoribonuclease activity.

Recent studies showed that Rai1 is a crucial component of the mRNA 5'-end-capping quality-control mechanism in yeast. The yeast genome encodes a weak homolog of Rai1, Ydr370C, but little is known about this protein. Here we report the crystal structures of Ydr370C from Kluyveromyces lactis and the first biochemical and functional studies on this protein. The overall structure of Ydr370C is similar to Rai1. Ydr370C has robust decapping activity on RNAs with unmethylated caps, but it has no detectable pyrophosphohydrolase activity. Unexpectedly, Ydr370C also possesses distributive, 5'-3' exoRNase activity, and we propose the name Dxo1 for this new eukaryotic enzyme with both decapping and exonuclease activities. Studies of yeast in which both Dxo1 and Rai1 are disrupted reveal that mRNAs with incomplete caps are produced even under normal growth conditions, in sharp contrast to current understanding of the capping process.

Chemical genetic profiling and characterization of small-molecule compounds that affect the biosynthesis of unsaturated fatty acids in Candida albicans.

The balance between saturated and unsaturated fatty acids plays a crucial role in determining the membrane fluidity. In the diploid fungal pathogen Candida albicans, the gene for fatty acid Delta9 desaturase, OLE1, is essential for viability. Using a reverse genetic approach, termed the fitness test, we identified a group of structurally related synthetic compounds that induce specific hypersensitivity of the OLE1(+/-) strain. Genetic repression of OLE1 and chemical inhibition by two selected compounds, ECC145 and ECC188, resulted in a marked decrease in the total unsaturated fatty acids and impaired hyphal development. The resulting auxotroph of both was suppressed by the exogenous monounsaturated fatty acids (16:1Delta9 and 18:1Delta9). These correlations suggest that both compounds affect the level of unsaturated fatty acids, likely by impairing Ole1p directly or indirectly. However, the residual levels of monounsaturated fatty acids (MUFAs) resulted from chemical inhibition were significantly higher than OLE1 repression, indicating even partial inhibition of MUFAs is sufficient to stop cellular proliferation. Although the essentiality of OLE1 was suppressed by MUFAs in vitro, we demonstrated that it was required for virulence in a murine model of systemic candidiasis even when the animals were supplemented with a high fat diet. Thus, the fungal fatty acid desaturase is an attractive antifungal drug target. Taking advantage of the inhibitors and the relevant conditional shut-off strains, we validated several chemical genetic interactions observed in the fitness test profiles that reveal novel genetic interactions between OLE1/unsaturated fatty acids and other cellular processes.

Candida albicans Spt23p controls the expression of the Ole1p Delta9 fatty acid desaturase and regulates unsaturated fatty acid biosynthesis.

In Saccharomyces cerevisiae the endoplasmic reticulum membrane proteins scSpt23p and scMga2p control the formation of unsaturated fatty acids by a mechanism that involves their release from the membrane by ubiquitin-mediated proteolysis. The resulting soluble polypeptides act as transcription activators that specifically control the expression of scOLE1, a gene that encodes scOle1p, a Delta9 fatty acid desaturase that forms cis-monounsaturated fatty acids (9Z-16:1 and 9Z-18:1) from saturated fatty acyl-CoA precursors. ScOle1p is the only long chain fatty acid desaturase in Saccharomyces and its membrane and storage lipids contain only saturated fatty acids and the monounsaturated products of that enzyme. Most other fungi, however, express multiple endoplasmic reticulum desaturases, including enzymes that form both mono- and polyunsaturated fatty acids. These typically include Delta12 and Delta15 enzymes that form the polyunsaturated species, 9Z,12Z-18:2, and 9Z,12Z,15Z-18:3, which are the most abundant fatty acids in membrane and storage lipids. An analysis of genomic DNA sequences shows that Candida albicans has a single homologue of the Saccharomyces scSPT23 and scMGA2 genes that we designate here as caSPT23. This study describes the characterization of the caSPT23 gene product and shows that it can repair the unsaturated fatty acid auxotrophy when it is expressed in a Saccharomyces scspt23Delta;scmga2Delta strain. In addition we show caSPT23 is essential for the expression of one of the two Delta9 desaturase homologues in Candida and potentially other functions associated with fatty acid metabolism.

Regulation of unsaturated fatty acid biosynthesis in Saccharomyces: the endoplasmic reticulum membrane protein, Mga2p, a transcription activator of the OLE1 gene, regulates the stability of the OLE1 mRNA through exosome-mediated mechanisms.

The Saccharomyces cerevisiae OLE1 gene encodes a membrane-bound Delta9 fatty-acid desaturase, whose expression is regulated through transcriptional and mRNA stability controls. In wild type cells grown on fatty acid-free medium, OLE1 mRNA has a half-life of 10 +/- 1.5 min (basal stability) that becomes highly unstable when cells are exposed to unsaturated fatty acids (regulated stability). Activation of OLE1 transcription is dependent on N-terminal fragments of two membrane proteins, Mga2p and Spt23p, that are proteolytically released from the membrane by a ubiquitin-mediated mechanism. Surprisingly, disruption of the MGA2 gene also reduces the half-life of the OLE1 transcript and abolishes fatty acid regulated instability. Disruption of its cognate, SPT23, has no effect on the half-life of the mRNA. Mga2p appears to have two distinct functions with respect to the OLE1 mRNA stability: a stabilizing effect in cells grown in fatty acid-free medium and a destabilizing function in cells that are exposed to unsaturated fatty acids. These functions are independent of OLE1 transcription and can confer basal and regulated stability on OLE1 mRNAs that are produced under the control of the unrelated GAL1 promoter. Expression of soluble, N-terminal fragments of Mga2p stabilize the transcript but do not confer fatty acid-regulated instability on the mRNA suggesting that the stabilizing functions of Mga2p do not require membrane processing and that modifications to the protein introduced during proteolysis may play a role in the destabilizing effect. An analysis of mutants that are defective in mRNA degradation indicate that the Mga2p-requiring control mechanism that regulates the fatty acid-mediated instability of the OLE1 transcript acts by activating exosomal 3' --> 5'-exonuclease degradation activity.

Maintenance and regulation of mRNA stability of the Saccharomyces cerevisiae OLE1 gene requires multiple elements within the transcript that act through translation-independent mechanisms.

The Saccharomyces cerevisiae OLE1 gene encodes a membrane-bound Delta-9 fatty acid desaturase, whose expression is regulated by unsaturated fatty acids through both transcriptional and mRNA stability controls. In fatty acid-free medium, the mRNA has a half-life of 10 +/- 1.5 min (basal stability) that drops to 2 +/- 1.5 min when cells are exposed to unsaturated fatty acids (regulated stability). A deletion analysis of elements within the transcript revealed that the sequences within the protein-coding region that encode transmembrane sequences and a part of the cytochrome b5 domain are essential for the basal stability of the transcript. Deletion of any of the three essential elements produced unstable transcripts and loss of regulated instability. By contrast, substitution of the 3'-untranslated region with that of the stable PGK1 gene did not affect the basal stability of the transcript and did not block regulated decay. Given that Ole1p is a membrane-bound protein whose activities are a major determinant of membrane fluidity, we asked whether membrane-associated translation of the protein was essential for basal and regulated stability. Insertion of stop codons within the transcript that blocked either translation of the entire protein or parts of the protein required for co-translation insertion of Ole1p had no effect. We conclude that the basal and regulated stability of the OLE1 transcript is resistant to the nonsense-mediated decay pathway and that the essential protein-encoding elements for basal stability act cooperatively as stabilizing sequences through RNA-protein interactions via a translation-independent mechanism.

Mga2p processing by hypoxia and unsaturated fatty acids in Saccharomyces cerevisiae: impact on LORE-dependent gene expression.

In Saccharomyces cerevisiae, OLE1 encodes a delta9 fatty acid desaturase, an enzyme that plays a critical role in maintaining the correct ratio of saturated to monounsaturated fatty acids in the cell membrane. Previous studies have demonstrated that (i) OLE1 expression is repressed by unsaturated fatty acids (UFAs) and induced by low oxygen tension, (ii) a component of this regulation is mediated through the same low oxygen response element (LORE) in the OLE1 promoter, and (iii) Mga2p is involved in LORE-dependent hypoxic induction of OLE1. We now report that LORE-CYC1 basal promoter-lacZ fusion reporter assays demonstrate that UFAs repress the reporter expression under hypoxic conditions in a dose-dependent manner via LORE. Electrophoretic mobility shift assays show that UFAs repress the hypoxia-induced complex formation with LORE. Studies with a construct encoding a truncated form of Mga2p support the hypothesis that both hypoxia and UFA signals affect the processing of Mga2p and the UFA repression of OLE1 hypoxic induction is mediated through Mga2p. Data from Western blot assays provide evidence that under normoxic conditions, Mga2p processing produces approximately equimolar levels of the membrane-bound and processed forms and is unaffected by UFAs. Hypoxic induction of OLE1, however, is associated with increased processing of the protein, resulting in an approximately fivefold increase in the soluble active form that is counteracted by exposure of the cells to unsaturated fatty acids. Data from this study suggest that the Mga2p-LORE interaction plays an important role in OLE1 expression under both normoxic and hypoxic conditions.