PDIP46 (DNA Polymerase Delta Interacting Protein 46) Is an Activating Factor for Human DNA Polymerase Delta

PDIP46 (SKAR, POLDIP3) was discovered through its interaction with the p50 subunit of human DNA polymerase δ (Pol δ). Its functions in DNA replication are unknown. PDIP46 associates with Pol δ in cell extracts both by immunochemical and protein separation methods, as well as by ChIP analyses. PDIP46 also interacts with PCNA via multiple copies of a novel PCNA binding motif, the APIMs (AlkB homologue-2 PCNA-Interacting Motif). Sites for both p50 and PCNA binding were mapped to the N-terminal region containing the APIMs. Functional assays for the effects of PDIP46 on Pol δ activity on singly primed ssM13 DNA templates revealed that it is a novel and potent activator of Pol δ. The effects of PDIP46 on Pol δ in primer extension, strand displacement and synthesis through simple hairpin structures reveal a mechanism where PDIP46 facilitates Pol δ4 synthesis through regions of secondary structure on complex templates. In addition, evidence was obtained that PDIP46 is also capable of exerting its effects by a direct interaction with Pol δ, independent of PCNA. Mutation of the Pol δ and PCNA binding region resulted in a loss of PDIP46 functions. These studies support the view that PDIP46 is a novel accessory protein for Pol δ that is involved in cellular DNA replication. This raises the possibility that altered expression of PDIP46 or its mutation may affect Pol δ functions in vivo, and thereby be a nexus for altered genomic stability

A Freeze Frame View of Vesicular Stomatitis Virus Transcription Defines a Minimal Length of RNA for 5′ Processing

The RNA synthesis machinery of vesicular stomatitis virus (VSV) comprises the genomic RNA encapsidated by the viral nucleocapsid protein (N) and associated with the RNA dependent RNA polymerase, the viral components of which are a large protein (L) and an accessory phosphoprotein (P). The 241 kDa L protein contains all the enzymatic activities necessary for synthesis of the viral mRNAs, including capping, cap methylation and polyadenylation. Those RNA processing reactions are intimately coordinated with nucleotide polymerization such that failure to cap results in termination of transcription and failure to methylate can result in hyper polyadenylation. The mRNA processing reactions thus serve as a critical check point in viral RNA synthesis which may control the synthesis of incorrectly modified RNAs. Here, we report the length at which viral transcripts first gain access to the capping machinery during synthesis. By reconstitution of transcription in vitro with highly purified recombinant polymerase and engineered templates in which we omitted sites for incorporation of UTP, we found that transcripts that were 30-nucleotides in length were uncapped, whereas those that were 31-nucleotides in length contained a cap structure. The minimal RNA length required for mRNA cap addition was also sufficient for methylation since the 31-nucleotide long transcripts were methylated at both ribose-2′-O and guanine-N-7 positions. This work provides insights into the spatial relationship between the active sites for the RNA dependent RNA polymerase and polyribonucleotidyltransferase responsible for capping of the viral RNA. We combine the present findings with our recently described electron microscopic structure of the VSV polymerase and propose a model of how the spatial arrangement of the capping activities of L may influence nucleotide polymerization

Protein Expression Redirects Vesicular Stomatitis Virus RNA Synthesis to Cytoplasmic Inclusions

Positive-strand and double-strand RNA viruses typically compartmentalize their replication machinery in infected cells. This is thought to shield viral RNA from detection by innate immune sensors and favor RNA synthesis. The picture for the non-segmented negative-strand (NNS) RNA viruses, however, is less clear. Working with vesicular stomatitis virus (VSV), a prototype of the NNS RNA viruses, we examined the location of the viral replication machinery and RNA synthesis in cells. By short-term labeling of viral RNA with 5′-bromouridine 5′-triphosphate (BrUTP), we demonstrate that primary mRNA synthesis occurs throughout the host cell cytoplasm. Protein synthesis results in the formation of inclusions that contain the viral RNA synthesis machinery and become the predominant sites of mRNA synthesis in the cell. Disruption of the microtubule network by treatment of cells with nocodazole leads to the accumulation of viral mRNA in discrete structures that decorate the surface of the inclusions. By pulse-chase analysis of the mRNA, we find that viral transcripts synthesized at the inclusions are transported away from the inclusions in a microtubule-dependent manner. Metabolic labeling of viral proteins revealed that inhibiting this transport step diminished the rate of translation. Collectively those data suggest that microtubule-dependent transport of viral mRNAs from inclusions facilitates their translation. Our experiments also show that during a VSV infection, protein synthesis is required to redirect viral RNA synthesis to intracytoplasmic inclusions. As viral RNA synthesis is initially unrestricted, we speculate that its subsequent confinement to inclusions might reflect a cellular response to infection

After COVID-19, all centenarians?

Con autorización del periódico para autores CSIC. El artículo apareció en la sección Agenda Social.Life expectancy for humans averaged around 35 years until the end of the 19th century. Since then, it has more than doubled, especially in Mediterranean countries where it now reaches almost 85 years. According to the World Economic Forum, in 2040 Spain would be the country with the highest life expectancy in the world (85.8 years) and Italy would rank fifth (84.5 years). The impact of the Covid-19 pandemic will affect these predictions as both countries have had an excess of deaths during 2020. However, both countries exemplify the increase of life expectancy during the last 120 years. The main factor promoting this increase in life expectancy is the development of antibiotics and vaccines that resulted in a marked decrease in bacterial and viral diseases that plagued our species throughout history. The current leading causes of death in the Western world, and especially in Europe, are dominated by coronary heart disease and cancer-related conditions. That is, diseases related to old age and poor eating habits in advanced societies. Nevertheless, infectious diseases are far from being eliminated as a threat, since bacteria and viruses continue to evolve and circumvent both natural and artificial barriers to infection.Peer reviewe

Opposing Effects of Inhibiting Cap Addition and Cap Methylation on Polyadenylation during Vesicular Stomatitis Virus mRNA Synthesis▿

The multifunctional large (L) polymerase protein of vesicular stomatitis virus (VSV) contains enzymatic activities essential for RNA synthesis, including mRNA cap addition and polyadenylation. We previously mapped amino acid residues G1154, T1157, H1227, and R1228, present within conserved region V (CRV) of L, as essential for mRNA cap addition. Here we show that alanine substitutions to these residues also affect 3′-end formation. Specifically, the cap-defective polymerases produced truncated transcripts that contained A-rich sequences at their 3′ termini and predominantly terminated within the first 500 nucleotides (nt) of the N gene. To examine how the cap-defective polymerases respond to an authentic VSV termination and reinitiation signal present at each gene junction, we reconstituted RNA synthesis using templates that contained genes inserted (I) at the leader-N gene junction. The I genes ranged in size from 382 to 1,098 nt and were typically transcribed into full-length uncapped transcripts. In addition to lacking a cap structure, the full-length I transcripts synthesized by the cap-defective polymerases lacked an authentic polyadenylate tail and instead contained 0 to 24 A residues. Moreover, the cap-defective polymerases were also unable to copy efficiently the downstream gene. Thus, single amino acid substitutions in CRV of L protein that inhibit cap addition also inhibit polyadenylation and sequential transcription of the genome. In contrast, an amino acid substitution, K1651A, in CRVI of L protein that completely inhibits cap methylation results in the hyperpolyadenylation of mRNA. This work reveals that inhibiting cap addition and cap methylation have opposing effects on polyadenylation during VSV mRNA synthesis and provides evidence in support of a link between correct 5′ cap formation and 3′ polyadenylation

Determination to a single nucleotide the length at which VSV mRNA gets capped.

<p>(A) An autoradiograph of a 6% polyacrylamide gel is shown indicating the products of the IVT reactions with recombinant VSV containing a 60-nt long non-essential gene that lacks sites for UTP incorporation except at positions 31, 32, 33, 34, 35, 36, 37 or 38 respectively. The reaction was performed in the absence of UTP, but containing [α-³²P]-GTP. The identity of the purified viruses used for the IVT reactions are shown at the top panel. The uncapped leader RNA and the different transcripts between 30 and 38 nt are indicated. Asterisks indicate the 1–2 nt longer than anticipated transcripts of the rVSV(A-)-33 and -34 viruses. (B) Recombinant VSV(A-)-30, -31, -33, -34, -35, -36 and -37 were used in <i>in vitro</i> transcription (IVT) reactions lacking UTP, but containing [α-³²P]-GTP. The products were analyzed on 6% polyacrylamide gel and visualized using a phosphorimager. The products of transcription were either untreated (-) or digested with TAP (+) which cleaves the cap-structure. The identity if the purified viruses used for the IVT reactions are shown at the top panel. The uncapped leader RNA and the different transcripts between 30 and 38 nt are indicated. A representative gel from two independent experiments is shown.</p

Capping occurs at a nascent RNA chain length of 31-nt.

<p><i>In vitro</i> transcription reactions were reconstituted with 5 µg of N-RNA template, 2 µg of purified P, and 4 µg of the indicated L protein in the absence of UTP, but in the presence of [α-³²P]-GTP. Purified RNA was analyzed on a 6% polyacrylamide gel and detected using a phosphorimager. The identity of the template and for the reactions used polymerase molecules are shown at the top the panel. The uncapped leader RNA and the different transcripts between 30 and 34 nt are indicated. Asterisks indicate the uncapped transcripts made by H1227A-L with the different templates. A representative gel from two independent experiments is shown.</p