Sedimentation profiles of U1-70K (Usp101p), U1H (Usp107p), U1J (Usp108) and U1L (Usp109p) complexes

<p><b>Copyright information:</b></p><p>Taken from "Proteomic analysis of the U1 snRNP of reveals three essential organism-specific proteins"</p><p></p><p>Nucleic Acids Research 2007;35(5):1391-1401.</p><p>Published online 30 Jan 2007</p><p>PMCID:PMC1865046.</p><p>© 2007 The Author(s).</p> () Protein extract (5 mg) of cells expressing U1-70K-TAP, U1H-TAP and U170-HA or U1J-TAP and U1-70K-HA or U1L-TAP and U1-10K-HA were separated on a 10–30% glycerol gradient. The gradient fractions () were precipitated with IgG antibodies and separated by SDS-PAGE, immunoblotted and probed with αTAP antibodies to determine the distribution of U1-70K-TAP, U1H-TAP, U1J-TAP and U1L-TAP, as indicated. () The gradient fractions 3–8 and 10–15 of a gradient-containing U1L-TAP and U1-70K-HA were pooled bound to IgG-Sepharose. The bound material was separated on SDS-PAGE, immunoblotted and probed with IgG and HA antibodies. An aliquot (50%) of the bound material was used to isolate RNA and hybridized with a labeled U1 probe to visualize U1 snRNA as indicated. Mo, pooled gradient fractions 10–15 of a protein extract from a wild-type strain. The pooled gradient fractions 3–8 from this gradient also showed no signals (not shown). The gradient was calibrated with small (30S) and large (50S) ribosomal subunits from

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

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

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

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