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PRPF8 defects cause missplicing in myeloid malignancies.

Kurtovic-Kozaric A, Przychodzen B, Singh J, Konarska MM, Clemente MJ, Otrock ZK, Nakashima M, Hsi ED, Yoshida K, Shiraishi Y, Chiba K, Tanaka H, Miyano S, Ogawa S, Boultwood J, Makishima H, Maciejewski JP, Padgett RA - Leukemia (2014)

Bottom Line: Fifty percent of PRPF8 mutant and del(17p) cases were found in AML and conveyed poor prognosis.Whole-RNA deep sequencing of primary cells from patients with PRPF8 abnormalities demonstrated consistent missplicing defects.In yeast models, homologous mutations introduced into Prp8 abrogated a block experimentally produced in the second step of the RNA splicing process, suggesting that the mutants have defects in proof-reading functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland, OH, USA.

ABSTRACT
Mutations of spliceosome components are common in myeloid neoplasms. One of the affected genes, PRPF8, encodes the most evolutionarily conserved spliceosomal protein. We identified either recurrent somatic PRPF8 mutations or hemizygous deletions in 15/447 and 24/450 cases, respectively. Fifty percent of PRPF8 mutant and del(17p) cases were found in AML and conveyed poor prognosis. PRPF8 defects correlated with increased myeloblasts and ring sideroblasts in cases without SF3B1 mutations. Knockdown of PRPF8 in K562 and CD34+ primary bone marrow cells increased proliferative capacity. Whole-RNA deep sequencing of primary cells from patients with PRPF8 abnormalities demonstrated consistent missplicing defects. In yeast models, homologous mutations introduced into Prp8 abrogated a block experimentally produced in the second step of the RNA splicing process, suggesting that the mutants have defects in proof-reading functions. In sum, the exploration of clinical and functional consequences suggests that PRPF8 is a novel leukemogenic gene in myeloid neoplasms with a distinct phenotype likely manifested through aberrant splicing.

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Related in: MedlinePlus

Yeast Saccharomyces cerevisiae in vivo Prp8 splicing assay(A) Schematic representation of the two-step splicing pathway (SS, splice site; BS, branch site). Briefly, two consecutive trans-esterifications involve three sites of the intron: in the first step, the BS attacks the 5′ SS, producing a lariat intermediate and the cleaved 5′ exon. In the second step, the 5′ exon attacks the 3′ SS, resulting in spliced mRNA and the lariat intron. Prp8 is required for both chemical steps and has been shown to regulate the conformational changes in the spliceosome that accompany the process. (B) Diagram of the ACT1-CUP1 reporter gene. The CUP1 gene allows growth in copper-containing media and is interrupted with an intron (originally from ACT1 gene) such that increased splicing results in increased resistance to copper. The intron tested here has been mutated so that the BS adenosine (bold) is placed 8 nucleotides away from exon 2, instead of 43 nucleotides as found in the wild type ACT1 intron. (C) Yeast growth on different copper concentrations (Y-axis). Different columns represent wt and disease-associated mutant alleles in the left panel, and control mutant alleles in the right panel. Amino acid designations are from yeast Prp8. Control p.R1753K is a first–step suppressor mutant, while p.V1870N and p.W1575R are second–step suppressor mutants. Mutations p.V1088D/I/N (hV1015), p.E1364K (hE1292), p.M1379I (hM1307I), p.D1670Y/H/N (hD1598Y) and p.H1947R (hH1875R) show a second-step suppressor phenotype, while p.N760P (hA687P), and p.G1822E (hG1750E) show a wild type phenotype.
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Figure 4: Yeast Saccharomyces cerevisiae in vivo Prp8 splicing assay(A) Schematic representation of the two-step splicing pathway (SS, splice site; BS, branch site). Briefly, two consecutive trans-esterifications involve three sites of the intron: in the first step, the BS attacks the 5′ SS, producing a lariat intermediate and the cleaved 5′ exon. In the second step, the 5′ exon attacks the 3′ SS, resulting in spliced mRNA and the lariat intron. Prp8 is required for both chemical steps and has been shown to regulate the conformational changes in the spliceosome that accompany the process. (B) Diagram of the ACT1-CUP1 reporter gene. The CUP1 gene allows growth in copper-containing media and is interrupted with an intron (originally from ACT1 gene) such that increased splicing results in increased resistance to copper. The intron tested here has been mutated so that the BS adenosine (bold) is placed 8 nucleotides away from exon 2, instead of 43 nucleotides as found in the wild type ACT1 intron. (C) Yeast growth on different copper concentrations (Y-axis). Different columns represent wt and disease-associated mutant alleles in the left panel, and control mutant alleles in the right panel. Amino acid designations are from yeast Prp8. Control p.R1753K is a first–step suppressor mutant, while p.V1870N and p.W1575R are second–step suppressor mutants. Mutations p.V1088D/I/N (hV1015), p.E1364K (hE1292), p.M1379I (hM1307I), p.D1670Y/H/N (hD1598Y) and p.H1947R (hH1875R) show a second-step suppressor phenotype, while p.N760P (hA687P), and p.G1822E (hG1750E) show a wild type phenotype.

Mentions: An important question arises as to whether the PRPF8 mutations confer an altered function or if they are loss of function alleles and thus merely recapitulate the haploinsufficiency of the deletion cases. Since the PRPF8 protein is exceptionally conserved between yeast and humans (Figure 1A), we created homologous mutations in yeast Prp8 to determine if the human disease-associated PRPF8 mutations cause splicing defects. Nine different yeast mutations: N760P (hA687P), V1088N (hV1015N), E1364K (hE1292K), M1379I (hM1307I), D1670Y/N (hD1598Y/N), G1822E (hG1750E), H1947R (h1875R), corresponding to the human somatic mutations were constructed and introduced into cells in which the chromosomal Prp8 gene was deleted, thus making the mutant Prp8 the only source of activity. In all cases, cell growth at all tested temperatures was indistinguishable from wild type suggesting that these mutations do not grossly affect function. To detect more subtle defects, we used a modification of the classical yeast splicing suppressor assay (Figure 4A, B). Briefly, we used a reporter plasmid that contains the ACT1–CUP1 reporter gene in which the ACT1 exon 1 and intron are inserted into the CUP1 gene. such that If splicing function is normal, the ACT1 intron is spliced out allowing the production of active CUP1 protein which confers a level of copper resistance to the cells that is proportional to the level of pre-mRNA splicing. If splicing is aberrant because of mutations in the ACT1 intron splice sites or Prp8 mutations, copper resistance is lowered. Previous studies have shown that certain splice site mutations in the ACT1 intron can be suppressed by second site mutations in splicing factors, particularly Prp8.34,49


PRPF8 defects cause missplicing in myeloid malignancies.

Kurtovic-Kozaric A, Przychodzen B, Singh J, Konarska MM, Clemente MJ, Otrock ZK, Nakashima M, Hsi ED, Yoshida K, Shiraishi Y, Chiba K, Tanaka H, Miyano S, Ogawa S, Boultwood J, Makishima H, Maciejewski JP, Padgett RA - Leukemia (2014)

Yeast Saccharomyces cerevisiae in vivo Prp8 splicing assay(A) Schematic representation of the two-step splicing pathway (SS, splice site; BS, branch site). Briefly, two consecutive trans-esterifications involve three sites of the intron: in the first step, the BS attacks the 5′ SS, producing a lariat intermediate and the cleaved 5′ exon. In the second step, the 5′ exon attacks the 3′ SS, resulting in spliced mRNA and the lariat intron. Prp8 is required for both chemical steps and has been shown to regulate the conformational changes in the spliceosome that accompany the process. (B) Diagram of the ACT1-CUP1 reporter gene. The CUP1 gene allows growth in copper-containing media and is interrupted with an intron (originally from ACT1 gene) such that increased splicing results in increased resistance to copper. The intron tested here has been mutated so that the BS adenosine (bold) is placed 8 nucleotides away from exon 2, instead of 43 nucleotides as found in the wild type ACT1 intron. (C) Yeast growth on different copper concentrations (Y-axis). Different columns represent wt and disease-associated mutant alleles in the left panel, and control mutant alleles in the right panel. Amino acid designations are from yeast Prp8. Control p.R1753K is a first–step suppressor mutant, while p.V1870N and p.W1575R are second–step suppressor mutants. Mutations p.V1088D/I/N (hV1015), p.E1364K (hE1292), p.M1379I (hM1307I), p.D1670Y/H/N (hD1598Y) and p.H1947R (hH1875R) show a second-step suppressor phenotype, while p.N760P (hA687P), and p.G1822E (hG1750E) show a wild type phenotype.
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Related In: Results  -  Collection

Show All Figures
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Figure 4: Yeast Saccharomyces cerevisiae in vivo Prp8 splicing assay(A) Schematic representation of the two-step splicing pathway (SS, splice site; BS, branch site). Briefly, two consecutive trans-esterifications involve three sites of the intron: in the first step, the BS attacks the 5′ SS, producing a lariat intermediate and the cleaved 5′ exon. In the second step, the 5′ exon attacks the 3′ SS, resulting in spliced mRNA and the lariat intron. Prp8 is required for both chemical steps and has been shown to regulate the conformational changes in the spliceosome that accompany the process. (B) Diagram of the ACT1-CUP1 reporter gene. The CUP1 gene allows growth in copper-containing media and is interrupted with an intron (originally from ACT1 gene) such that increased splicing results in increased resistance to copper. The intron tested here has been mutated so that the BS adenosine (bold) is placed 8 nucleotides away from exon 2, instead of 43 nucleotides as found in the wild type ACT1 intron. (C) Yeast growth on different copper concentrations (Y-axis). Different columns represent wt and disease-associated mutant alleles in the left panel, and control mutant alleles in the right panel. Amino acid designations are from yeast Prp8. Control p.R1753K is a first–step suppressor mutant, while p.V1870N and p.W1575R are second–step suppressor mutants. Mutations p.V1088D/I/N (hV1015), p.E1364K (hE1292), p.M1379I (hM1307I), p.D1670Y/H/N (hD1598Y) and p.H1947R (hH1875R) show a second-step suppressor phenotype, while p.N760P (hA687P), and p.G1822E (hG1750E) show a wild type phenotype.
Mentions: An important question arises as to whether the PRPF8 mutations confer an altered function or if they are loss of function alleles and thus merely recapitulate the haploinsufficiency of the deletion cases. Since the PRPF8 protein is exceptionally conserved between yeast and humans (Figure 1A), we created homologous mutations in yeast Prp8 to determine if the human disease-associated PRPF8 mutations cause splicing defects. Nine different yeast mutations: N760P (hA687P), V1088N (hV1015N), E1364K (hE1292K), M1379I (hM1307I), D1670Y/N (hD1598Y/N), G1822E (hG1750E), H1947R (h1875R), corresponding to the human somatic mutations were constructed and introduced into cells in which the chromosomal Prp8 gene was deleted, thus making the mutant Prp8 the only source of activity. In all cases, cell growth at all tested temperatures was indistinguishable from wild type suggesting that these mutations do not grossly affect function. To detect more subtle defects, we used a modification of the classical yeast splicing suppressor assay (Figure 4A, B). Briefly, we used a reporter plasmid that contains the ACT1–CUP1 reporter gene in which the ACT1 exon 1 and intron are inserted into the CUP1 gene. such that If splicing function is normal, the ACT1 intron is spliced out allowing the production of active CUP1 protein which confers a level of copper resistance to the cells that is proportional to the level of pre-mRNA splicing. If splicing is aberrant because of mutations in the ACT1 intron splice sites or Prp8 mutations, copper resistance is lowered. Previous studies have shown that certain splice site mutations in the ACT1 intron can be suppressed by second site mutations in splicing factors, particularly Prp8.34,49

Bottom Line: Fifty percent of PRPF8 mutant and del(17p) cases were found in AML and conveyed poor prognosis.Whole-RNA deep sequencing of primary cells from patients with PRPF8 abnormalities demonstrated consistent missplicing defects.In yeast models, homologous mutations introduced into Prp8 abrogated a block experimentally produced in the second step of the RNA splicing process, suggesting that the mutants have defects in proof-reading functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland, OH, USA.

ABSTRACT
Mutations of spliceosome components are common in myeloid neoplasms. One of the affected genes, PRPF8, encodes the most evolutionarily conserved spliceosomal protein. We identified either recurrent somatic PRPF8 mutations or hemizygous deletions in 15/447 and 24/450 cases, respectively. Fifty percent of PRPF8 mutant and del(17p) cases were found in AML and conveyed poor prognosis. PRPF8 defects correlated with increased myeloblasts and ring sideroblasts in cases without SF3B1 mutations. Knockdown of PRPF8 in K562 and CD34+ primary bone marrow cells increased proliferative capacity. Whole-RNA deep sequencing of primary cells from patients with PRPF8 abnormalities demonstrated consistent missplicing defects. In yeast models, homologous mutations introduced into Prp8 abrogated a block experimentally produced in the second step of the RNA splicing process, suggesting that the mutants have defects in proof-reading functions. In sum, the exploration of clinical and functional consequences suggests that PRPF8 is a novel leukemogenic gene in myeloid neoplasms with a distinct phenotype likely manifested through aberrant splicing.

Show MeSH
Related in: MedlinePlus