The N-terminus of Prp1 (Prp6/U5-102 K) is essential for spliceosome activation in vivo

The spliceosomal protein Prp1 (Prp6/U5-102 K) is necessary for the integrity of pre-catalytic spliceosomal complexes. We have identified a novel regulatory function for Prp1. Expression of mutations in the N-terminus of Prp1 leads to the accumulation of pre-catalytic spliceosomal complexes containing the five snRNAs U1, U2, U5 and U4/U6 and pre-mRNAs. The mutations in the N-terminus, which prevent splicing to occur, include in vitro and in vivo identified phosphorylation sites of Prp4 kinase. These sites are highly conserved in the human ortholog U5-102 K. The results presented here demonstrate that structural integrity of the N-terminus is required to mediate a splicing event, but is not necessary for the assembly of spliceosomes

Rrn7 protein, an RNA polymerase I transcription factor, is required for RNA polymerase II-dependent transcription directed by core promoters with a HomolD box sequence

The region in promoters that specifies the transcription machinery is called the core promoter, displaying core promoter elements (CPE) necessary for establishment of a preinitiation complex and the initiation of transcription. A classical CPE is the TATA box. In fission yeast, Schizosaccharomyces pombe, a new CPE, called HomolD box, was discovered. Collectively, 141 ribosomal protein genes encoding the full set of 79 different ribosomal proteins and more than 60 other housekeeping genes display a HomolD box in the core promoter. Here, we show that transcription directed by the HomolD box requires the RNA polymerase II machinery, including the general transcription factors. Most intriguingly, however, we identify, by DNA affinity purification, Rrn7 as the protein binding to the HomolD box. Rrn7 is an evolutionary conserved member of the RNA polymerase I machinery involved in transcription initiation of core ribosomal DNA promoters. ChIP shows that Rrn7 cross-links to a ribosomal prote

Prp4 kinase grants the license to splice: control of weak splice sites during spliceosome activation

The genome of the fission yeast Schizosaccharomyces pombe encodes 17 kinases that are essential for cell growth. These include the cell-cycle regulator Cdc2, as well as several kinases that coordinate cell growth, polarity, and morphogenesis during the cell cycle. In this study, we further characterized another of these essential kinases, Prp4, and showed that the splicing of many introns is dependent on Prp4 kinase activity. For detailed characterization, we chose the genes res1 and ppk8, each of which contains one intron of typical size and position. Splicing of the res1 intron was dependent on Prp4 kinase activity, whereas splicing of the ppk8 intron was not. Extensive mutational analyses of the 5' splice site of both genes revealed that proper transient interaction with the 5' end of snRNA U1 governs the dependence of splicing on Prp4 kinase activity. Proper transient interaction between the branch sequence and snRNA U2 was also important. Therefore, the Prp4 kinase is required for recognition and efficient splicing of introns displaying weak exon1/5' splice sites and weak branch sequences.We thank the DAAD (Deutscher Akademischer Austausch Dienst, German Academic Exchange Service) for their support of this project as part of the Spanish–German exchange program,which enabled DE to work in José Ayté’s laboratoryat the Universitat Pompeu Fabra (Barcelona, Spain). The Spanish Ministry of Science and Innovation (BFU2012-31939), PLAN E and FEDER to JA and a Georg-Christoph-Lichtenberg scholarship to AR, provided by the federal state of Niedersachsen (Germany)

Prp4_as2 kinase and its inhibition with 1NM-PP1 in fission yeast.

<p>(A) A strain with the genotype <i>h</i>^<i>−s</i><i>prp4-as2</i> was grown at 30°C to early log-phase. The inhibitor 1NM-PP1 was then added to the culture medium (0 hours, arrow, <b>↓</b>) at a final concentration of 10 μM. Growth of the culture was monitored by counting the number of cells/mL (squares) relative to a culture growing in the absence of inhibitor (circles). The error bars indicate standard deviation. (B) Percentage of septated cells during growth in the absence (-Inh) and presence (+Inh) of inhibitor. Bars show the mean value of three independent repetitions (n = 3) and error bars indicate the standard deviation. A two-tailed t-test was performed to check whether the number of septated cells differs significantly without and with inhibition of the kinase (* = p < 0.05; ** = p < 0.01; *** = p < 0.001). (C) DNA content analysis in C of <i>prp4-as2</i> cells immediately before (-Inh) and at the indicated times after the addition of 1NM-PP1 (+). (D) RT-PCR analyses of RNA prepared at the indicated times after the addition of inhibitor (+Inh). RNA was also extracted from cells grown in the absence of inhibitor (-Inh). Specific primers were used to detect <i>res2</i>, <i>rpl29</i>, <i>res1</i>, <i>tbp1</i>-III, <i>cdc2</i> I+II and <i>cdc2</i> III+IV RNAs. Roman numerals indicate the intron numbers contained within the amplicons. The numbers on the right side of the image represent the sizes of the RT-PCR fragments (bp). Asterisks indicate the expected positions of fragments if the introns between the indicated primer pairs are not spliced out. H₂O, negative control without template. The numbers on the left side of the image represent the sizes of the DNA fragments (bp). M, DNA size marker.</p

The Prp4 kinase dependence of the <i>res1</i> intron can be changed by mutations in the exon1/5’ splice site.

<p>(A) Schematic representation of the <i>res1</i>^<i>+</i> and <i>res1’</i> genes. The <i>res1’</i> gene was integrated by homologous recombination into the <i>leu1</i> locus. Because Res1 is essential for growth, all strains containing the <i>res1’</i> gene also contain <i>res1</i>^<i>+</i>. (B) Proposed base-pairing between the <i>res1</i>^<i>+</i> exon1/5’ SS region and snRNA U1. Ψ indicates the pseudouridine 3 nucleotides from the 5’ end of snRNA U1. Numbering of the exon1/5’ SS region is indicated. (C–I) RT-PCR analysis in the absence (-Inh) and presence (+Inh) of inhibitor at the indicated times. H₂O, negative control without template. The scheme on the left side of the image shows the details of the interactions between the exon1/5’ SS region and snRNA U1. Small letters indicate the mutations in the <i>res1’</i> exon1/5’ SS; the corresponding alleles were named as indicated. |, Watson-Crick base-pairing; +, wobble base-pairing G-U; Ψ, Pseudouridine; ϕ, wobble base-pairing Ψ-A. Asterisks indicate the expected position of fragments if the introns are or are not spliced out. The numbers on the left side of the image represent the sizes of the DNA fragments (bp). M, DNA size marker. C compares the endogenous <i>res1</i>^<i>+</i> with the integrated <i>res1’</i>.</p

Whole-genome splicing profile of a fission yeast strain expressing Prp4_as2 kinase.

<p>(A) The frequency histograms assign the number of introns found for each calculated Relative Splicing Efficiency Index (RSEI) in the absence (- Inhibitor) of 1NM-PP1 or after 30 and 60 min in the presence of inhibitor. The bin size is 0.05. Bars with negative RSEI values display Prp4-dependent introns (1008 introns) while bars with positive RSEI values represent Prp4-independent introns (2557 introns). The dashed line marks the 0 value. (B) Similar size distributions of Prp4-independent and -dependent introns. (C) A hypothetical example of a fission yeast pre-mRNA. Consensus sequences of the 5’ SS, branch sequence with branch point A (bp, arrow), and 3’ SS are shown. These consensus sequences do not differ between Prp4-dependent and–independent introns. The sequence logos were generated using WebLogo [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005768#pgen.1005768.ref074" target="_blank">74</a>]. (D) Splicing of the introns of <i>rpb5</i>, <i>tbp1</i>, and <i>mrp17</i> monitored by RT-PCR using specific primers, as indicated in the schemes above the images, in the absence of inhibitor (-Inh) or after 10 and 30 minutes in the presence of inhibitor (+Inh). Asterisks indicate the expected position of fragments if the introns are not spliced out. RSEI below the images was obtained from cells collected after 30 min in the presence of inhibitor. Roman numerals indicate the 5’→3’ order of introns. The numbers on the left side of the image represent the sizes of the DNA fragments (bp). M, DNA size marker.</p

Point mutation in the third position of the branch sequence converts a Prp4 kinase-independent intron into a kinase-dependent intron.

<p>(A) Proposed base-pairing between the <i>res1</i> intron branch sequence CUAAC and snRNA U2. Ψ indicates the pseudouridine 39 nucleotides from the 5’ end of snRNA U2, which is suggested to base-pair with the A at position 3 of the branch sequence. (B) <i>res1’-A</i> and <i>res1’-2A</i>: RT-PCR analysis in the absence (-Inh) and presence (+Inh) of inhibitor at the indicated times. (C) <i>res1’-B</i> and <i>res1’-2B</i>. (D) <i>res1’-C</i> and <i>res1’-2C</i>. (E) <i>res1’-D</i> and <i>res1’-2D</i> (F) <i>res1’-E</i> and <i>res1’-2E</i>. H₂O, negative control without template. The scheme on the left side of the images show the details of the interactions between exon1/5’ SS and snRNA U1 and between the branch sequence and snRNA U2. Small letters indicate the mutations in exon1/5’ SS and the branch sequence; the corresponding alleles were named as indicated. |, Watson-Crick base-pairing; Ψ, Pseudouridine; ϕ, wobble base-pairing Ψ-A. Asterisks indicate the expected position of fragments if the introns are or are not spliced out. The numbers on the left side of the image represent the sizes of the DNA fragments (bp). M, DNA size marker.</p

SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface.

COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a new recently emerged sarbecovirus. This virus uses the human ACE2 enzyme as receptor for cell entry, recognizing it with the receptor binding domain (RBD) of the S1 subunit of the viral spike protein. We present the use of phage display to select anti-SARS-CoV-2 spike antibodies from the human naïve antibody gene libraries HAL9/10 and subsequent identification of 309 unique fully human antibodies against S1. 17 antibodies are binding to the RBD, showing inhibition of spike binding to cells expressing ACE2 as scFv-Fc and neutralize active SARS-CoV-2 virus infection of VeroE6 cells. The antibody STE73-2E9 is showing neutralization of active SARS-CoV-2 as IgG and is binding to the ACE2-RBD interface. Thus, universal libraries from healthy human donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovering patients in a pandemic situation