A simple, non-radioactive DNA fingerprinting method for identifying patrilines in honeybee colonies

Primers were derived flanking a microsatellite motif of the cloned Z-locus. The PCR product of the Z-locus was variable in size and up to four alleles were found in a sample of 11 workers within one colony. Using the combination of three loci, the Z, the Q (both linked to the sex locus) and a royal jelly protein gene (RJP57-1) we were able to discriminate five patrilines in the 11 worker sample. Using the well established microsatellite technology, however, seven and six patrilines could be identified. The technique may enable laboratories which lack an isotope facility and equipped with only a PCR thermocycler and agarose gel apparatus to study the polyandrous mating system of the honeybee in a variety of different contexts. © Inra/DIB/AGIB/Elsevier, Pari

Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis

Molnupiravir is an orally available antiviral drug candidate currently in phase III trials for the treatment of patients with COVID-19. Molnupiravir increases the frequency of viral RNA mutations and impairs SARS-CoV-2 replication in animal models and in humans. Here, we establish the molecular mechanisms underlying molnupiravir-induced RNA mutagenesis by the viral RNA-dependent RNA polymerase (RdRp). Biochemical assays show that the RdRp uses the active form of molnupiravir, β-d-$N^4$-hydroxycytidine (NHC) triphosphate, as a substrate instead of cytidine triphosphate or uridine triphosphate. When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp–RNA complexes that contain mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism probably applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir

Dynamic Contacts of U2, RES, Cwc25, Prp8 and Prp45 Proteins with the Pre-mRNA Branch-Site and 3' Splice Site during Catalytic Activation and Step 1 Catalysis in Yeast Spliceosomes

<div><p>Little is known about contacts in the spliceosome between proteins and intron nucleotides surrounding the pre-mRNA branch-site and their dynamics during splicing. We investigated protein-pre-mRNA interactions by UV-induced crosslinking of purified yeast B^act spliceosomes formed on site-specifically labeled pre-mRNA, and analyzed their changes after conversion to catalytically-activated B* and step 1 C complexes, using a purified splicing system. Contacts between nucleotides upstream and downstream of the branch-site and the U2 SF3a/b proteins Prp9, Prp11, Hsh49, Cus1 and Hsh155 were detected, demonstrating that these interactions are evolutionarily conserved. The RES proteins Pml1 and Bud13 were shown to contact the intron downstream of the branch-site. A comparison of the B^act crosslinking pattern versus that of B* and C complexes revealed that U2 and RES protein interactions with the intron are dynamic. Upon step 1 catalysis, Cwc25 contacts with the branch-site region, and enhanced crosslinks of Prp8 and Prp45 with nucleotides surrounding the branch-site were observed. Cwc25’s step 1 promoting activity was not dependent on its interaction with pre-mRNA, indicating it acts via protein-protein interactions. These studies provide important insights into the spliceosome's protein-pre-mRNA network and reveal novel RNP remodeling events during the catalytic activation of the spliceosome and step 1 of splicing.</p></div

Interaction of Prp46 and Prp45 with the region around the 3’SS during catalytic activation and step 1 catalysis.

<p>(A) Autoradiography of portions of 2D gels comprising total proteins of purified, UV-irradiated and RNase digested B^act, B* and C complexes, respectively. The asterisks indicate contaminant crosslinked proteins. (B) Schematic representation of site-specifically labeled pre-mRNAs as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005539#pgen.1005539.g002" target="_blank">Fig 2</a>. B^actΔPrp2 spliceosomes were assembled on pre-mRNAs site-specifically labeled 5’ of the different G nucleotides as shown, in splicing extracts of a yeast <i>prp2-1</i> strain carrying Prp46 tagged with the TAP-tag and were purified according to protocol 2 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005539#pgen.1005539.s011" target="_blank">S1 Text</a>). Spliceosomes were then UV irradiated, digested with RNase T1, and subjected to immunoprecipitation with IgG Sepharose. Immunoprecipitates were analyzed by SDS-PAGE and subsequent western blotting as above (upper panel). The western blot shows a band of the expected size of Prp46-TAP. The autoradiography of the membrane is shown in the lower panel and the RNA fragments crosslinked to Prp46-TAP are marked by an arrow. (C) as in (B) B^act spliceosomes were assembled on the pre-mRNA site-specifically labeled 5’ of the G nucleotide at position 496, in splicing extracts of a yeast <i>prp2-1</i> strain carrying Prp45 tagged with the TAP-tag. Upon addition of Prp2/Spp2 and Cwc25, B* and C complexes were obtained. The western blot shows a band of the expected size of Prp45-TAP. The RNA fragment crosslinked to Prp45-TAP is marked by an arrow.</p

Site-specific UV crosslinking of U2 SF3a/b and RES complex proteins to the intron region around the branch-site in the yeast spliceosomal B^{act ΔPrp2} complex.

<p>(A). Schematic representation of site-specifically labeled pre-mRNAs carrying a single ³²P-labeled phosphate 5’ of the guanosines shown in green. The RNA fragments remaining after digestion with RNase T1 are indicated by a box below the sequence. Spliceosomes were assembled on site-specifically labeled pre-mRNAs in splicing extracts of a yeast <i>prp2-1</i> strain carrying proteins tagged with the TAP-tag at their C termini. B^{act ΔPrp2} complexes were purified according to protocol 2 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005539#pgen.1005539.s011" target="_blank">S1 Text</a>). (B). Purified B^{act ΔPrp2} complexes containing TAP-tagged U2 SF3a/b proteins were UV irradiated (+ lanes) or non-irradiated (–lanes). All samples were then digested with RNase T1 and subjected to immunoprecipitation with IgG Sepharose. Immunoprecipitates were analyzed by SDS-PAGE and subsequent western blotting with peroxidase anti-peroxidase (PAP) complex antibody (upper panel). The western blot shows bands of the expected size of the U2 proteins indicated (note that the TAP-tag increases the size of a protein by ca. 21 kDa). The autoradiography of the membrane is shown in the lower panel. (C) As in (B), except that purified B^actΔPrp2 complexes containing TAP-tagged RES complex proteins were used. The arrows indicate ³²P-labeled RNA fragments crosslinked to the respective proteins.</p

2D gel electrophoresis of affinity-purified yeast spliceosomal B^{act ΔPrp2} complexes.

<p>(A) Schematic representation of the 3’-region-labeled actin pre-mRNA which was used to assemble spliceosomal complexes for 2D gel electrophoresis. The 3’ portion of the pre-mRNA body-labeled with ³²P-UTP is shown in black and includes the guanosine at position 426 of the intron up to the end of the 3’ exon. The unlabeled pre-mRNA is shown in gray. (B) B^act complexes were purified according to protocol 1 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005539#pgen.1005539.s011" target="_blank">S1 Text</a>). Total proteins of purified, non-irradiated (–UV) and RNase-digested B^act complexes were separated electrophoretically by 2D gel electrophoresis and then stained with RuBPS [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005539#pgen.1005539.ref023" target="_blank">23</a>]. The directions of the first-dimension and second-dimension electrophoreses are shown at the top and on the left. In the first-dimension of gel electrophoresis, proteins are separated by charge while in the second-dimension they are separated according to their molecular weight. The proteins observed in the predominant spots were cut from the gel and analyzed by mass spectrometry, and the proteins identified are indicated. Two predominant spots corresponding to the contaminant proteins Xrn1 and Hrb1 are indicated; additional predominant spots corresponding to contaminant proteins are labeled with asterisks, from top to bottom: Prp5, Scp160, Kre33, Sup35, Bfr1 and Nop1. Those corresponding to RNases and MS2-MBP are also indicated. (C) Autoradiography of the 2D gel comprising total proteins of purified, UV-irradiated (+UV) and RNase digested B^act complexes. The circles indicate the position of the RuBPS stained spots shown in (B). Radioactive spots corresponding to proteins that were not further characterized in this work are indicated by a dot, from top to bottom: Cwc22, Cwc2/Cwc27, Isy1.</p

Summary of site-specific UV crosslinking of proteins to the intron region around the branch-site in the B^act complex and their dynamics following the conversion of B^act into the B* complex (i.e., catalytically activated) and during the subsequent conversion of the latter into the C complex (i.e., the step 1 spliceosome).

<p>Schematic representation of the secondary-structure model of U2/U6/pre-mRNA interactions in the B^act complex. The branch point A is indicated by a red bold letter. Sites in the pre-mRNA's intron crosslinked to the U2-SF3a (grey), U2-SF3b (green), RES complex proteins [(shades of purple, represented schematically according to [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005539#pgen.1005539.ref020" target="_blank">20</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005539#pgen.1005539.ref038" target="_blank">38</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005539#pgen.1005539.ref040" target="_blank">40</a>]], Prp8, Cwc25 and Prp45 are indicated by the number of the site-specifically labeled guanosines. The regions of site-specific UV-crosslinking are also summarized on the Tables on the right. Changes in crosslinking yields upon conversion of B^act to B* to C are highlighted by changes in color intensities.</p