A BBP–Mud2p heterodimer mediates branchpoint recognition and influences splicing substrate abundance in budding yeast

The 3′ end of mammalian introns is marked by the branchpoint binding protein, SF1, and the U2AF65-U2AF35 heterodimer bound at an adjacent sequence. Baker's yeast has equivalent proteins, branchpoint binding protein (BBP) (SF1) and Mud2p (U2AF65), but lacks an obvious U2AF35 homolog, leaving open the question of whether another protein substitutes during spliceosome assembly. Gel filtration, affinity selection and mass spectrometry were used to show that rather than a U2AF65/U2AF35-like heterodimer, Mud2p forms a complex with BBP without a third (U2AF35-like) factor. Using mutants of MUD2 and BBP, we show that the BBP–Mud2p complex bridges partner-specific Prp39p, Mer1p, Clf1p and Smy2p two-hybrid interactions. In addition to inhibiting Mud2p association, the bbpΔ56 mutation impairs splicing, enhances pre-mRNA release from the nucleus, and similar to a mud2::KAN knockout, suppresses a lethal sub2::KAN mutation. Unexpectedly, rather than exacerbating bbpΔ56, the mud2::KAN mutation partially suppresses a pre-mRNA accumulation defect observed with bbpΔ56. We propose that a BBP–Mud2p heterodimer binds as a unit to the branchpoint in vivo and serves as a target for the Sub2p-DExD/H-box ATPase and for other splicing factors during spliceosome assembly. In addition, our results suggest the possibility that the Mud2p may enhance the turnover of pre-mRNA with impaired BBP-branchpoint association

Structure–function analysis and genetic interactions of the yeast branchpoint binding protein Msl5

Saccharomyces cerevisiae Msl5 (branchpoint binding protein) orchestrates spliceosome assembly by binding the branchpoint sequence 5′-UACUAAC and establishing cross intron-bridging interactions with other components of the splicing machinery. Reciprocal tandem affinity purifications verify that Msl5 exists in vivo as a heterodimer with Mud2 and that the Msl5–Mud2 complex is associated with the U1 snRNP. By gauging the ability of mutants of Msl5 to complement msl5Δ, we find that the Mud2-binding (amino acids 35–54) and putative Prp40-binding (PPxY100) elements of the Msl5 N-terminal domain are inessential, as are the C-terminal proline-rich domain (amino acids 382–476) and two zinc-binding CxxCxxxxHxxxxC motifs (amino acids 273–286 and 299–312). A subset of conserved branchpoint RNA-binding amino acids in the central KH-QUA2 domain (amino acids 146–269) are essential pairwise (Ile198–Arg190; Leu256–Leu259) or in trios (Leu169–Arg172–Leu176), whereas other pairs of RNA-binding residues are dispensable. We used our collection of viable Msl5 mutants to interrogate synthetic genetic interactions, in cis between the inessential structural elements of the Msl5 polypeptide and in trans between Msl5 and yeast splicing factors (Mud2, Nam8 and Tgs1) that are optional for vegetative growth. The results suggest a network of important but functionally buffered protein–protein and protein–RNA interactions between the Mud2–Msl5 complex at the branchpoint and the U1 snRNP at the 5′ splice site

Functional mammalian spliceosomal complex E contains SMN complex proteins in addition to U1 and U2 snRNPs

Copyright @ 2011 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.Spliceosomes remove introns from primary gene transcripts. They assemble de novo on each intron through a series of steps that involve the incorporation of five snRNP particles and multiple non-snRNP proteins. In mammals, all the intermediate complexes have been characterized on one transcript (MINX), with the exception of the very first, complex E. We have purified this complex by two independent procedures using antibodies to either U1-A or PRPF40A proteins, which are known to associate at an early stage of assembly. We demonstrate that the purified complexes are functional in splicing using commitment assays. These complexes contain components expected to be in the E complex and a number of previously unrecognized factors, including survival of motor neurons (SMN) and proteins of the SMN-associated complex. Depletion of the SMN complex proteins from nuclear extracts inhibits formation of the E complex and causes non-productive complexes to accumulate. This suggests that the SMN complex stabilizes the association of U1 and U2 snRNPs with pre-mRNA. In addition, the antibody to PRPF40A precipitated U2 snRNPs from nuclear extracts, indicating that PRPF40A associates with U2 snRNPs

Ксантогранулематозный пиелонефрит в сочетании с замещающим липоматозом почки. КТ-диагностика. Cлучай из практики

Xanthogranulomatous pyelonephritis is a relatively rare form of chronic pyelonephritis. It is characterized by combination of destructive and proliferative processes with proliferation in the kidney of granulomatous tissue with a large number of lipid-containing macrophages, which are called xanthomous or "foamy" cells. Replacement kidney lipomatosis is also a rare disease that is characterized by proliferation of adipose tissue of the kidney sinus and perirenal adipose tissue with pronounced atrophy of the renal parenchyma. This is the result of renal atrophy, chronic inflammation, nephrolithiasis or long-term current hydronephrosis with the proliferation of adipose tissue in the renal sinus and paranephric fiber. The article presents a case of combining the above-mentioned pathological processes in a patient of the 93-ies. According to clinical manifestations and examination data, xanthogranulomatous pyelonephritis can occur under the guise of pionephrosis, carbuncle, abscess. Deservedly this disease is called "great imitator", because today there are no clear diagnostic criteria, and the diagnosis is established, as a rule, in the postoperative period.Ксантогранулематозный пиелонефрит – относительно редкая форма хронического пиелонефрита. Для него характерно сочетание деструктивного и пролиферативного процессов с разрастанием в почке грануле матозной ткани с большим количеством липидсодержащих макрофагов, которые именуются ксантом ными, или “пенистыми”, клетками. Замещающий липоматоз почки также является редким заболеванием, которое характеризуется пролиферацией жировой ткани почечного синуса и периренальной жировой ткани с выраженной атрофией почечной паренхимы. Это результат почечной атрофии, хронического воспаления, нефролитиаза или длительно текущего гидронефроза с пролиферацией жировой ткани в почечном синусе и паранефральной клетчатке. В статье представлено наблюдение сочетания вышеназванных патологических процессов у пациента 93 лет. По клиническим проявлениям и данным обследования ксантогранулематозный пиелонефрит может протекать под видом пионефроза, карбункула, абсцесса. Заслуженно это заболевание называют “great imitator” (“великий притворщик”), поскольку на сегодняшний день нет четких диагностических критериев, и диагноз устанавливается, как правило, в послеоперационном периоде

Characterisation of the biflavonoid hinokiflavone as a premRNA splicing modulator that inhibits SENP

We have identified the plant biflavonoid hinokiflavone as an inhibitor of splicing in vitro and modulator of alternative splicing in cells. Chemical synthesis confirms hinokiflavone is the active molecule. Hinokiflavone inhibits splicing in vitro by blocking spliceosome assembly, leading to accumulation of the A complex. Cells treated with hinokiflavone show altered subnuclear organization specifically of splicing factors required for A complex formation, which relocalize together with SUMO1 and SUMO2 into enlarged nuclear speckles. Hinokiflavone increases protein SUMOylation levels, both in in vitro splicing reactions and in cells. Hinokiflavone also inhibited a purified, E. coli expressed SUMO protease, SENP1, in vitro, indicating the increase in SUMOylated proteins results primarily from inhibition of de-SUMOylation. Using a quantitative proteomics assay we identified many SUMO2 sites whose levels increased in cells following hinokiflavone treatment, with the major targets including 6 proteins that are components of the U2 snRNP and required for A complex formation

Современный подход к компьютерно-томографической диагностике аденокарциномы легкого

The classification of tumors of the lung, pleura, thymus and heart was published by the World Health Organization (WHO) in 2015. It presents a completely different approach to adenocarcinoma compared to the 2004 WHO classification.Adenocarcinoma is the most common histological type of lung cancer.The interdisciplinary classification is based on consensus among oncologists, thoracic surgeons, pulmonologists, pathologists, molecular biologists, radiologists, radiologists and identifies a wide range of adenocarcinoma types and subtypes with varying prognosis and treatment. They are accompanied by a variety of manifestations and features on computed tomography, which usually correlate with histopathological findings, highlighting one of the key roles of the radiologist in the diagnosis and treatment of such patients.The aim of the work is to familiarize radiologists with the WHO 2015 classification, terminology and computed tomographic diagnostic criteria for various types of lung adenocarcinoma.Классификация опухолей легкого, плевры, тимуса и сердца опубликована Всемирной организации здравоохранения (ВОЗ) в 2015 г. В ней представлен совершенно иной подход к аденокарциноме по сравнению с классификацией ВОЗ 2004 г.Аденокарцинома – наиболее распространенный гистологический тип рака легких.Междисциплинарная классификация основана на консенсусе между онкологами, торакальными хирургами, пульмонологами, патологами, молекулярными биологами, рентгенологами, радиологами и определяет широкий спектр типов аденокарциномы и подтипов с различным прогнозом и лечением. Они сопровождаются разнообразными проявлениями и особенностями при компьютерной томографии, которые обычно коррелируют с гистопатологическими данными, подчеркивая одну из ключевых ролей врача-рентгенолога в диагностике и лечении таких пациентов.Целью работы является ознакомление врачей-рентгенологов с классификацией ВОЗ 2015, терминологией и компьютерно-томографическими диагностическими критериями различных типов аденокарциномы легкого.

COVID-19. Вопросы диагностики и лечения поражения легких

Aim. This publication is devoted to a brief review of the unresolved questions of medical imaging and treatment of COVID-19.Materials and methods. The analysis of publications for the first quarter of 2020 on radiation diagnosis and treatment of COVID-19 with an emphasis on morpho-radiological comparisons was performed.Results. It was established that CT can be used for the initial “sorting” of patients in determining the need and conditions of hospitalization, but in the future, often leads to disagreement among pulmonologists and radiologists in assessing the dynamics of the development of the disease. In most patients with COVID-19, the pathomorphological and radiological patterns indicate the formation of the acute phase of diffuse alveolar damage, transforming into organizing pneumonia with typical CT manifestations. However, to date there are no established opinions on the management of patients with corresponding lung lesions.Conclusion. The literature data on the peculiarities of the pathomorphological and radiological patterns of COVID-19 COVID-19 justify the advisability of using corticosteroids in the treatment of this disease.Цель исследования: краткий обзор нерешенных вопросов лучевой диагностики и лечения COVID-19.Материал и методы. Проведен анализ публикаций за I квартал 2020 г. по лучевой диагностике и лечению COVID-19 с акцентом на морфорентгенологические сопоставления.Результаты. Установлено, что КТ может быть использована для первичной “сортировки” пациентов при определении необходимости и условий госпитализации, но в дальнейшем зачастую приводит к разногласию пульмонологов и рентгенологов в оценке динамики развития заболевания. У большинства пациентов с COVID-19 патоморфологическая и рентгенологическая картина свидетельствует о формировании острой фазы диффузного альвеолярного повреждения, трансформирующегося в организующую пневмонию с типичными КТ-проявлениями. Однако до настоящего времени нет устоявшихся мнений по тактике ведения пациентов с подобным поражением легких.Выводы. Приведенные данные литературы об особенностях патоморфологической и рентгенологической картины COVID-19 обосновывают целесообразность применения кортикостероидов в лечении данного заболевания

The U1, U2 and U5 snRNAs crosslink to the 5′ exon during yeast pre-mRNA splicing

Activation of pre-messenger RNA (pre-mRNA) splicing requires 5′ splice site recognition by U1 small nuclear RNA (snRNA), which is replaced by U5 and U6 snRNA. Here we use crosslinking to investigate snRNA interactions with the 5′ exon adjacent to the 5′ splice site, prior to the first step of splicing. U1 snRNA was found to interact with four different 5′ exon positions using one specific sequence adjacent to U1 snRNA helix 1. This novel interaction of U1 we propose occurs before U1-5′ splice site base pairing. In contrast, U5 snRNA interactions with the 5′ exon of the pre-mRNA progressively shift towards the 5′ end of U5 loop 1 as the crosslinking group is placed further from the 5′ splice site, with only interactions closest to the 5′ splice site persisting to the 5′ exon intermediate and the second step of splicing. A novel yeast U2 snRNA interaction with the 5′ exon was also identified, which is ATP dependent and requires U2-branchpoint interaction. This study provides insight into the nature and timing of snRNA interactions required for 5′ splice site recognition prior to the first step of pre-mRNA splicing

Determinants of Nam8-dependent splicing of meiotic pre-mRNAs

Nam8, a component of yeast U1 snRNP, is optional for mitotic growth but required during meiosis, because Nam8 collaborates with Mer1 to promote splicing of essential meiotic mRNAs AMA1, MER2 and MER3. Here, we identify SPO22 and PCH2 as novel targets of Nam8-dependent meiotic splicing. Whereas SPO22 splicing is co-dependent on Mer1, PCH2 is not. The SPO22 intron has a non-consensus 5′ splice site (5′SS) that dictates its Nam8/Mer1-dependence. SPO22 splicing relies on Mer1 recognition, via its KH domain, of an intronic enhancer 5′-AYACCCUY. Mutagenesis of KH and the enhancer highlights Arg214 and Gln243 and the CCC triplet as essential for Mer1 activity. The Nam8-dependent PCH2 pre-mRNA has a consensus 5′SS and lacks a Mer1 enhancer. For PCH2, a long 5′ exon and a non-consensus intron branchpoint dictate Nam8-dependence. Our results implicate Nam8 in two distinct meiotic splicing regulons. Nam8 is composed of three RRM domains, flanked by N-terminal leader and C-terminal tail segments. The leader, tail and RRM1 are dispensable for splicing meiotic targets and unnecessary for vegetative Nam8 function in multiple synthetic lethal genetic backgrounds. Nam8 activity is enfeebled by alanine mutations in the putative RNA binding sites of the RRM2 and RRM3 domains