IDENTIFICATION OF NOVEL PUTATIVE SNORNAS IN AML PATIENT SAMPLES
(Abstract release date: 05/19/16)
EHA Library. Gerloff D. 06/09/16; 132429; E880
Dr. Dennis Gerloff
Contributions
Contributions
Abstract
Abstract: E880
Type: Eposter Presentation
Background
Small nucleolar RNAs (snoRNAs) are a large group of non-coding RNAs. Mostly snoRNAs are located in introns of host genes. They are highly abundant in eukaryotes and are divided into two major families, box C/D and box H/ACA snoRNAs, based of common sequence motifs and structural features. The box C/D snoRNAs carry the conserved boxes C (RUGAUGA, R=purine) and D (CUGA) near their 5' and 3' ends, respectively. The H/ACA box snoRNAs consist of two hairpins and two short single-stranded regions, which contain the H box (ANANNA) and the ACA box. snoRNAs guide small nucleolar ribonucleoproteins (snoRNPs) to complementary regions of ribosomal (rRNA) or small nuclear RNAs (snRNA), where the snoRNP complexes catalyze ribosomal modifications. Recent studies showed the relevance of snoRNAs for the pathogenesis of cancer.
Aims
Identification and analysis of unknown snoRNAs in AML.
Methods
To understand the role of snoRNAs in leukemogenesis, we analyzed the snoRNA expression pattern of 63 AML patients, registered in a recent clinical trial (ClinicalTrials.gov NCT00915252). RNA was isolated with Qiagen miRNeasy Kit. Librarys for small RNA specific next generation sequencing (NGS) were prepared with TruSeq Small RNASample Prep Kit and size separation of 40-200 nucleotides. NGS was performed on an Illumina HiScanSQ, using 50 cycles of single read sequencing. Sequencing data were processed with Cutadapt, mapped with Bowtie 2 to the human genome (hg19). Abundant regions were detected with Bedtools genomeCoverageBed and were annotated using snoRNABase (LBME), DASHR, UCSC snoRNA and repeatmasker track. For further analysis reads of discovered regions were calculated as reads per million (RPM). Regions without annotation were analyzed for C/D box motifs to discover putative C/D box snoRNAs.
Results
Next generation sequencing data revealed that 229 of the 269 known C/D box snoRNAs (85%) were expressed in one or more patient samples. For the H/ACA box snoRNAs we could identify 90 of 112 (80%). In addition we found 1396 mapped regions, which could not be annotated as known small noncoding RNAs. In silico analysis identified 90 putative C/D box snoRNAs. All of these putative snoRNAs contain the box C/D motifs and were mostly located in introns of known genes.In further analysis we compared clinical data and snoRNA expression pattern. Here we found that expression of the known snoRNAs clustered with specific risk groups. High levels of snoRNA expression are associated with intermediate molecular risk grouped patients (269 snoRNAs, p≤0.05) and a poor response to chemotherapy (101 snoRNAs, p≤0.05). Likewise, increased expression of putative snoRNAs was found in patients with an intermediate molecular risk (51 snoRNAs, p≤0.05) and in patients with a bad response to chemotherapy (20 snoRNAs, p≤0.05).To investigate putative snoRNAs during myeloid differentiation we treated HL60 cells with 1µM all-transretinoic acid (ATRA) and analyzed their expression (d0 vs. d6). In the HL60 cells we could recover 83 putative snoRNAs out of 90 we found in the patient samples. The data show that 45 putative snoRNAs were downregulated (<0.7) while 17 putative snoRNAs were upregulated (>1.3) during myeloid differentiation. To analyze the binding of 8 putative snoRNA candidates to the snRNP complex we performed NOP58 and FBL specific RNA immunoprecipitation (RIP) in Kasumi-1 cells. Here, we could show an enriched binding of 2 putative snoRNAs to FBL and NOP58.
Conclusion
In summary, our data show that snoRNAs may contribute to neoplastic transformation. Further we identified novel unknown snoRNAs, which will be further analyzed.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia
Type: Eposter Presentation
Background
Small nucleolar RNAs (snoRNAs) are a large group of non-coding RNAs. Mostly snoRNAs are located in introns of host genes. They are highly abundant in eukaryotes and are divided into two major families, box C/D and box H/ACA snoRNAs, based of common sequence motifs and structural features. The box C/D snoRNAs carry the conserved boxes C (RUGAUGA, R=purine) and D (CUGA) near their 5' and 3' ends, respectively. The H/ACA box snoRNAs consist of two hairpins and two short single-stranded regions, which contain the H box (ANANNA) and the ACA box. snoRNAs guide small nucleolar ribonucleoproteins (snoRNPs) to complementary regions of ribosomal (rRNA) or small nuclear RNAs (snRNA), where the snoRNP complexes catalyze ribosomal modifications. Recent studies showed the relevance of snoRNAs for the pathogenesis of cancer.
Aims
Identification and analysis of unknown snoRNAs in AML.
Methods
To understand the role of snoRNAs in leukemogenesis, we analyzed the snoRNA expression pattern of 63 AML patients, registered in a recent clinical trial (ClinicalTrials.gov NCT00915252). RNA was isolated with Qiagen miRNeasy Kit. Librarys for small RNA specific next generation sequencing (NGS) were prepared with TruSeq Small RNASample Prep Kit and size separation of 40-200 nucleotides. NGS was performed on an Illumina HiScanSQ, using 50 cycles of single read sequencing. Sequencing data were processed with Cutadapt, mapped with Bowtie 2 to the human genome (hg19). Abundant regions were detected with Bedtools genomeCoverageBed and were annotated using snoRNABase (LBME), DASHR, UCSC snoRNA and repeatmasker track. For further analysis reads of discovered regions were calculated as reads per million (RPM). Regions without annotation were analyzed for C/D box motifs to discover putative C/D box snoRNAs.
Results
Next generation sequencing data revealed that 229 of the 269 known C/D box snoRNAs (85%) were expressed in one or more patient samples. For the H/ACA box snoRNAs we could identify 90 of 112 (80%). In addition we found 1396 mapped regions, which could not be annotated as known small noncoding RNAs. In silico analysis identified 90 putative C/D box snoRNAs. All of these putative snoRNAs contain the box C/D motifs and were mostly located in introns of known genes.In further analysis we compared clinical data and snoRNA expression pattern. Here we found that expression of the known snoRNAs clustered with specific risk groups. High levels of snoRNA expression are associated with intermediate molecular risk grouped patients (269 snoRNAs, p≤0.05) and a poor response to chemotherapy (101 snoRNAs, p≤0.05). Likewise, increased expression of putative snoRNAs was found in patients with an intermediate molecular risk (51 snoRNAs, p≤0.05) and in patients with a bad response to chemotherapy (20 snoRNAs, p≤0.05).To investigate putative snoRNAs during myeloid differentiation we treated HL60 cells with 1µM all-transretinoic acid (ATRA) and analyzed their expression (d0 vs. d6). In the HL60 cells we could recover 83 putative snoRNAs out of 90 we found in the patient samples. The data show that 45 putative snoRNAs were downregulated (<0.7) while 17 putative snoRNAs were upregulated (>1.3) during myeloid differentiation. To analyze the binding of 8 putative snoRNA candidates to the snRNP complex we performed NOP58 and FBL specific RNA immunoprecipitation (RIP) in Kasumi-1 cells. Here, we could show an enriched binding of 2 putative snoRNAs to FBL and NOP58.
Conclusion
In summary, our data show that snoRNAs may contribute to neoplastic transformation. Further we identified novel unknown snoRNAs, which will be further analyzed.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia
Abstract: E880
Type: Eposter Presentation
Background
Small nucleolar RNAs (snoRNAs) are a large group of non-coding RNAs. Mostly snoRNAs are located in introns of host genes. They are highly abundant in eukaryotes and are divided into two major families, box C/D and box H/ACA snoRNAs, based of common sequence motifs and structural features. The box C/D snoRNAs carry the conserved boxes C (RUGAUGA, R=purine) and D (CUGA) near their 5' and 3' ends, respectively. The H/ACA box snoRNAs consist of two hairpins and two short single-stranded regions, which contain the H box (ANANNA) and the ACA box. snoRNAs guide small nucleolar ribonucleoproteins (snoRNPs) to complementary regions of ribosomal (rRNA) or small nuclear RNAs (snRNA), where the snoRNP complexes catalyze ribosomal modifications. Recent studies showed the relevance of snoRNAs for the pathogenesis of cancer.
Aims
Identification and analysis of unknown snoRNAs in AML.
Methods
To understand the role of snoRNAs in leukemogenesis, we analyzed the snoRNA expression pattern of 63 AML patients, registered in a recent clinical trial (ClinicalTrials.gov NCT00915252). RNA was isolated with Qiagen miRNeasy Kit. Librarys for small RNA specific next generation sequencing (NGS) were prepared with TruSeq Small RNASample Prep Kit and size separation of 40-200 nucleotides. NGS was performed on an Illumina HiScanSQ, using 50 cycles of single read sequencing. Sequencing data were processed with Cutadapt, mapped with Bowtie 2 to the human genome (hg19). Abundant regions were detected with Bedtools genomeCoverageBed and were annotated using snoRNABase (LBME), DASHR, UCSC snoRNA and repeatmasker track. For further analysis reads of discovered regions were calculated as reads per million (RPM). Regions without annotation were analyzed for C/D box motifs to discover putative C/D box snoRNAs.
Results
Next generation sequencing data revealed that 229 of the 269 known C/D box snoRNAs (85%) were expressed in one or more patient samples. For the H/ACA box snoRNAs we could identify 90 of 112 (80%). In addition we found 1396 mapped regions, which could not be annotated as known small noncoding RNAs. In silico analysis identified 90 putative C/D box snoRNAs. All of these putative snoRNAs contain the box C/D motifs and were mostly located in introns of known genes.In further analysis we compared clinical data and snoRNA expression pattern. Here we found that expression of the known snoRNAs clustered with specific risk groups. High levels of snoRNA expression are associated with intermediate molecular risk grouped patients (269 snoRNAs, p≤0.05) and a poor response to chemotherapy (101 snoRNAs, p≤0.05). Likewise, increased expression of putative snoRNAs was found in patients with an intermediate molecular risk (51 snoRNAs, p≤0.05) and in patients with a bad response to chemotherapy (20 snoRNAs, p≤0.05).To investigate putative snoRNAs during myeloid differentiation we treated HL60 cells with 1µM all-transretinoic acid (ATRA) and analyzed their expression (d0 vs. d6). In the HL60 cells we could recover 83 putative snoRNAs out of 90 we found in the patient samples. The data show that 45 putative snoRNAs were downregulated (<0.7) while 17 putative snoRNAs were upregulated (>1.3) during myeloid differentiation. To analyze the binding of 8 putative snoRNA candidates to the snRNP complex we performed NOP58 and FBL specific RNA immunoprecipitation (RIP) in Kasumi-1 cells. Here, we could show an enriched binding of 2 putative snoRNAs to FBL and NOP58.
Conclusion
In summary, our data show that snoRNAs may contribute to neoplastic transformation. Further we identified novel unknown snoRNAs, which will be further analyzed.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia
Type: Eposter Presentation
Background
Small nucleolar RNAs (snoRNAs) are a large group of non-coding RNAs. Mostly snoRNAs are located in introns of host genes. They are highly abundant in eukaryotes and are divided into two major families, box C/D and box H/ACA snoRNAs, based of common sequence motifs and structural features. The box C/D snoRNAs carry the conserved boxes C (RUGAUGA, R=purine) and D (CUGA) near their 5' and 3' ends, respectively. The H/ACA box snoRNAs consist of two hairpins and two short single-stranded regions, which contain the H box (ANANNA) and the ACA box. snoRNAs guide small nucleolar ribonucleoproteins (snoRNPs) to complementary regions of ribosomal (rRNA) or small nuclear RNAs (snRNA), where the snoRNP complexes catalyze ribosomal modifications. Recent studies showed the relevance of snoRNAs for the pathogenesis of cancer.
Aims
Identification and analysis of unknown snoRNAs in AML.
Methods
To understand the role of snoRNAs in leukemogenesis, we analyzed the snoRNA expression pattern of 63 AML patients, registered in a recent clinical trial (ClinicalTrials.gov NCT00915252). RNA was isolated with Qiagen miRNeasy Kit. Librarys for small RNA specific next generation sequencing (NGS) were prepared with TruSeq Small RNASample Prep Kit and size separation of 40-200 nucleotides. NGS was performed on an Illumina HiScanSQ, using 50 cycles of single read sequencing. Sequencing data were processed with Cutadapt, mapped with Bowtie 2 to the human genome (hg19). Abundant regions were detected with Bedtools genomeCoverageBed and were annotated using snoRNABase (LBME), DASHR, UCSC snoRNA and repeatmasker track. For further analysis reads of discovered regions were calculated as reads per million (RPM). Regions without annotation were analyzed for C/D box motifs to discover putative C/D box snoRNAs.
Results
Next generation sequencing data revealed that 229 of the 269 known C/D box snoRNAs (85%) were expressed in one or more patient samples. For the H/ACA box snoRNAs we could identify 90 of 112 (80%). In addition we found 1396 mapped regions, which could not be annotated as known small noncoding RNAs. In silico analysis identified 90 putative C/D box snoRNAs. All of these putative snoRNAs contain the box C/D motifs and were mostly located in introns of known genes.In further analysis we compared clinical data and snoRNA expression pattern. Here we found that expression of the known snoRNAs clustered with specific risk groups. High levels of snoRNA expression are associated with intermediate molecular risk grouped patients (269 snoRNAs, p≤0.05) and a poor response to chemotherapy (101 snoRNAs, p≤0.05). Likewise, increased expression of putative snoRNAs was found in patients with an intermediate molecular risk (51 snoRNAs, p≤0.05) and in patients with a bad response to chemotherapy (20 snoRNAs, p≤0.05).To investigate putative snoRNAs during myeloid differentiation we treated HL60 cells with 1µM all-transretinoic acid (ATRA) and analyzed their expression (d0 vs. d6). In the HL60 cells we could recover 83 putative snoRNAs out of 90 we found in the patient samples. The data show that 45 putative snoRNAs were downregulated (<0.7) while 17 putative snoRNAs were upregulated (>1.3) during myeloid differentiation. To analyze the binding of 8 putative snoRNA candidates to the snRNP complex we performed NOP58 and FBL specific RNA immunoprecipitation (RIP) in Kasumi-1 cells. Here, we could show an enriched binding of 2 putative snoRNAs to FBL and NOP58.
Conclusion
In summary, our data show that snoRNAs may contribute to neoplastic transformation. Further we identified novel unknown snoRNAs, which will be further analyzed.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia
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