Isolierung und Charakterisierung von Suppressoren des prp1 Gens, einer regulatorischen Komponente des prä-katalytischen Spleißosoms der Spalthefe

Prp1 (U5-102K/Prp6) is a highly conserved spliceosomal protein necessary for the structural integrity of pre-catalytic spliceosomes. The protein consists of multiple HAT (half a TPR) repeats preceded by an N-terminal domain showing no similarity with known motifs. We show here that mutations within the N-terminus lead to the accumulation of pre-catalytic spliceosomal particles which consist of snRNPs U1, U2, U5, U4/U6 and contain unspliced pre-mRNA. These results demonstrate that structural integrity of the N-terminus is required to mediate a splicing event, but is not necessary for the assembly of spliceosomes. The temperature sensitive allele prp1-127ts containing a point mutation in HAT8 was used to isolate six extragenic suppressors. Two extragenic suppressors, spp101-1 and spp102-1, were further analysed: spp102 encodes Spp42/Prp8 (U5-220K), a protein which has been shown to act as a central organizer during activation of a spliceosome; spp101 encodes Lin1 (U5-52K). Lin1 is not stably associated with Prp1 suggesting transient interaction between these proteins. In a strain expressing prp1-127ts the steady state level of the mutant Prp1G705D protein is visibly lower when compared with Prp1wt in a wild-type strain at 35 ºC. The expression of the mutant Lin1D167N protein in the suppressor strain (spp101-1/lin1-1 prp1-127ts) does not lead to the stabilization of Prp1G705D at 35 °C suggesting that the mechanism of suppression is not due to the stabilization of Prp1 by Lin1. The mutant Prp1G705D still able to associate with pre-catalytic spliceosomes prevents their activation at the restrictive temperature. Expression of the suppressor gene spp101-1/lin1-1, however, allows activation of those pre-catalytic spliceosomes associated with Prp1G705D. Therefore, it is conceivable that Lin1D167N improves the interactions of other spliceosomal proteins with Prp1G705D thus operating as a chaperone.Prp1 (U5-102K/Prp6) ist ein hochkonserviertes spleißosomales Protein welches für die strukturelle Integrität des prä-katalytischen Spleißosoms essentiell ist. Das Protein besteht aus multiplen Wiederholungen eines HAT Sequenzmotivs, denen eine N-terminale Domäne vorangeht, die keine Ähnlichkeit mit bekannten Sequenzmotiven aufweist. Mutationen in der N-terminalen Domäne führen zur Akkumulation von prä-katalytischen spleißosomalen Partikeln, die aus den snRNPs U1, U2, U5 und U4/U6, sowie ungespleißter prä-mRNA zusammengesetzt sind. Dieses Ergebnis zeigt, daß die strukturelle Integrität der N-terminalen Domäne erforderlich ist um ein Speißereignis zu vermitteln, aber nicht für den Zusammenbau des Spleißosoms erforderlich ist. Das temperatur-sensitive Allel prp1-127ts, das eine Punktmutation im achten HAT-Motiv enthält, wurde dazu verwendet sechs extragene Suppressoren zu isolieren. Davon wurden zwei, spp101-1 und spp102-1 näher untersucht: spp102-1 kodiert für Spp42/Prp8 (U5-220K), einem Protein, das bei der Aktivierung des Spleißosoms von besonderer Bedeutung ist; spp101-1 kodiert für Lin1 (U5-52K). Lin1 ist nicht stabil an Prp1 gebunden, was auf eine transiente Interaktion zwischen beiden Proteinen hinweist. In einem Stamm der das prp1-127ts Allel exprimiert ist die Konzentration des mutierten Prp1G705D Proteins viel geringer als die Konzentration von Prp1wt in einem wildtyp Stamm, der bei 35 °C gewachsen ist. Die Expression des mutierten Lin1D167N Proteins im Suppressor Stamm (spp101-1/lin1-1 prp1-127ts) führt nicht zur Stabilisierung von Prp1G705D, was darauf hinweist, daß die Suppression nicht über eine von Lin1 vermittelte Stabilisierung von Prp1G705D erfolgt. Das mutierte Prp1G705D Protein kann noch immer mit dem prä-katalytischen Spleißosom assoziieren und so seine Aktivierung bei der restriktiven Temperatur verhindern. Dennoch ermöglicht die Expression des Suppressorgens spp101-1/lin1-1 die Aktivierung des mit Prp1G705D assoziierten prä-katalytischen Spleißosoms. Daher erscheint es möglich, daß Lin1D167N die Interaktion zwischen Prp1G705D und anderen spleißosomalen Proteinen fördert, d.h. als Chaperon funktioniert

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

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)

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

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

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