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Strand-specific RNA sequencing in Plasmodium falciparum malaria identifies developmentally regulated long non-coding RNA and circular RNA.

Broadbent KM, Broadbent JC, Ribacke U, Wirth D, Rinn JL, Sabeti PC - BMC Genomics (2015)

Bottom Line: This approach enabled the annotation of over one thousand lncRNA transcript models and their comprehensive global analysis: coding prediction, periodicity, stage-specificity, correlation, GC content, length, location relative to annotated transcripts, and splicing.As compared to neighboring mRNAs, the expression of antisense-sense pairs was significantly anti-correlated during blood stage development, indicating transcriptional interference.We also validated that P. falciparum produces circRNAs, which is notable given the lack of RNA interference in the organism, and discovered that a highly expressed, five-exon antisense RNA is poised to regulate P. falciparum gametocyte development 1 (PfGDV1), a gene required for early sexual commitment events.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA. k8broadbent@post.harvard.edu.

ABSTRACT

Background: The human malaria parasite Plasmodium falciparum has a complex and multi-stage life cycle that requires extensive and precise gene regulation to allow invasion and hijacking of host cells, transmission, and immune escape. To date, the regulatory elements orchestrating these critical parasite processes remain largely unknown. Yet it is becoming increasingly clear that long non-coding RNAs (lncRNAs) could represent a missing regulatory layer across a broad range of organisms.

Results: To investigate the regulatory capacity of lncRNA in P. falciparum, we harvested fifteen samples from two time-courses. Our sample set profiled 56 h of P. falciparum blood stage development. We then developed and validated strand-specific, non-polyA-selected RNA sequencing methods, and pursued the first assembly of P. falciparum strand-specific transcript structures from RNA sequencing data. This approach enabled the annotation of over one thousand lncRNA transcript models and their comprehensive global analysis: coding prediction, periodicity, stage-specificity, correlation, GC content, length, location relative to annotated transcripts, and splicing. We validated the complete splicing structure of three lncRNAs with compelling properties. Non-polyA-selected deep sequencing also enabled the prediction of hundreds of intriguing P. falciparum circular RNAs, six of which we validated experimentally.

Conclusions: We found that a subset of lncRNAs, including all subtelomeric lncRNAs, strongly peaked in expression during invasion. By contrast, antisense transcript levels significantly dropped during invasion. As compared to neighboring mRNAs, the expression of antisense-sense pairs was significantly anti-correlated during blood stage development, indicating transcriptional interference. We also validated that P. falciparum produces circRNAs, which is notable given the lack of RNA interference in the organism, and discovered that a highly expressed, five-exon antisense RNA is poised to regulate P. falciparum gametocyte development 1 (PfGDV1), a gene required for early sexual commitment events.

No MeSH data available.


Related in: MedlinePlus

Notable lncRNAs include multi-exonic and telomere-associated transcripts. (A)/(B) Multi-exonic antisense transcripts span an apicoplast RNA methyltransferase precursor [PlasmoDB:Pf3D7_0218300] and an ETRAMP [PlasmoDB:Pf3D7_0936100] gene, respectively. Annotated gene models are shown in dark green and dark blue, and assembled transcript models are shown in light green and light blue. Reads from each 56-hour time course sample mapping to the (−) strand are shown below each horizontal axis in light green, while reads mapping to the (+) strand are shown above each horizontal axis in light blue. Intron reads are shown in purple. Uniqueness of 100mers is plotted in red as a mappability track. (C)/(D) Pearson correlation between the Pf3D7_0218300 sense-antisense pair and ETRAMP sense-antisense pair during the 56-hour time course was 0.20 and −0.50, respectively. Notably, Pf3D7_0218300 and ETRAMP antisense transcript levels dropped during parasite invasion, while sense transcript levels did not. Expression is plotted in units of log2(FPKM + 1). (E) Multi-exonic lncRNAs are encoded in the PfGDV1 region on chromosome nine, antisense to PfGDV1 and between PfGDV1 and GEXP22. Refer to (A)/(B) for a description of tracks. (F)/(G) Pearson correlation between the PfGDV1 sense-antisense pair was 0.96, while Pearson correlation between the divergent intergenic lncRNA and GEXP22 pair was 0.46 during the 56-hour time course. Expression is plotted in units of log2(FPKM + 1). (H) As we have previously described, the telomere-associated repetitive element (TARE) 2–3 region transcribes a family of lncRNA-TAREs, with transcription always proceeding towards the telomere [41]. For example, lncRNA-TARE-2 L is transcribed on the left arm of chromosome two. Pf3D7_0200100 is a subtelomeric upsB-type PfEMP1-encoding var gene. Boundaries of the telomere, TAREs 1–5, and Rep20 are shown in purple. See (A)/(B) for a further description of tracks. (I) Plotting the expression level of 22 lncRNA-TARE family members showed that lncRNA-TARE expression was co-regulated, with maximal firing coinciding with parasite invasion. Expression is plotted in units of log2(FPKM + 1). (J) Pearson correlation between lncRNA-TARE-2 L and the neighboring PfEMP1-encoding var gene was −0.09 during the 56-hour time course. Expression is plotted in units of log2(FPKM + 1)
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Fig4: Notable lncRNAs include multi-exonic and telomere-associated transcripts. (A)/(B) Multi-exonic antisense transcripts span an apicoplast RNA methyltransferase precursor [PlasmoDB:Pf3D7_0218300] and an ETRAMP [PlasmoDB:Pf3D7_0936100] gene, respectively. Annotated gene models are shown in dark green and dark blue, and assembled transcript models are shown in light green and light blue. Reads from each 56-hour time course sample mapping to the (−) strand are shown below each horizontal axis in light green, while reads mapping to the (+) strand are shown above each horizontal axis in light blue. Intron reads are shown in purple. Uniqueness of 100mers is plotted in red as a mappability track. (C)/(D) Pearson correlation between the Pf3D7_0218300 sense-antisense pair and ETRAMP sense-antisense pair during the 56-hour time course was 0.20 and −0.50, respectively. Notably, Pf3D7_0218300 and ETRAMP antisense transcript levels dropped during parasite invasion, while sense transcript levels did not. Expression is plotted in units of log2(FPKM + 1). (E) Multi-exonic lncRNAs are encoded in the PfGDV1 region on chromosome nine, antisense to PfGDV1 and between PfGDV1 and GEXP22. Refer to (A)/(B) for a description of tracks. (F)/(G) Pearson correlation between the PfGDV1 sense-antisense pair was 0.96, while Pearson correlation between the divergent intergenic lncRNA and GEXP22 pair was 0.46 during the 56-hour time course. Expression is plotted in units of log2(FPKM + 1). (H) As we have previously described, the telomere-associated repetitive element (TARE) 2–3 region transcribes a family of lncRNA-TAREs, with transcription always proceeding towards the telomere [41]. For example, lncRNA-TARE-2 L is transcribed on the left arm of chromosome two. Pf3D7_0200100 is a subtelomeric upsB-type PfEMP1-encoding var gene. Boundaries of the telomere, TAREs 1–5, and Rep20 are shown in purple. See (A)/(B) for a further description of tracks. (I) Plotting the expression level of 22 lncRNA-TARE family members showed that lncRNA-TARE expression was co-regulated, with maximal firing coinciding with parasite invasion. Expression is plotted in units of log2(FPKM + 1). (J) Pearson correlation between lncRNA-TARE-2 L and the neighboring PfEMP1-encoding var gene was −0.09 during the 56-hour time course. Expression is plotted in units of log2(FPKM + 1)

Mentions: Based on the diverse characteristics examined above, we searched for transcripts with exceptional properties. For example, we found that a putative Apicoplast RNA methyltransferase precursor [PlasmoDB:Pf3D7_0218300] and an Early Transcribed Membrane Protein [ETRAMP; PlasmoDB:Pf3D7_0936100] transcribe multi-exonic antisense RNAs across their full gene bodies [Fig. 4A and B]. Expression of the Apicoplast RNA methyltransferase precursor sense-antisense pair was not particularly correlated (ρ = .20), while expression of the ETRAMP sense-antisense pair was moderately anti-correlated (ρ = −.50) [Fig. 4C and D]. Interestingly, ETRAMP antisense transcription was substantially higher than ETRAMP sense transcription, reaching a maximum FPKM of 550 in early stages. This was the highest expression level observed for predicted P. falciparum antisense RNAs at any stage. Both the Apicoplast RNA methyltransferase precursor and ETRAMP antisense RNAs also demonstrated the 48 hpi expression drop phenomenon, though their sense partners did not exhibit this pattern.Fig. 4


Strand-specific RNA sequencing in Plasmodium falciparum malaria identifies developmentally regulated long non-coding RNA and circular RNA.

Broadbent KM, Broadbent JC, Ribacke U, Wirth D, Rinn JL, Sabeti PC - BMC Genomics (2015)

Notable lncRNAs include multi-exonic and telomere-associated transcripts. (A)/(B) Multi-exonic antisense transcripts span an apicoplast RNA methyltransferase precursor [PlasmoDB:Pf3D7_0218300] and an ETRAMP [PlasmoDB:Pf3D7_0936100] gene, respectively. Annotated gene models are shown in dark green and dark blue, and assembled transcript models are shown in light green and light blue. Reads from each 56-hour time course sample mapping to the (−) strand are shown below each horizontal axis in light green, while reads mapping to the (+) strand are shown above each horizontal axis in light blue. Intron reads are shown in purple. Uniqueness of 100mers is plotted in red as a mappability track. (C)/(D) Pearson correlation between the Pf3D7_0218300 sense-antisense pair and ETRAMP sense-antisense pair during the 56-hour time course was 0.20 and −0.50, respectively. Notably, Pf3D7_0218300 and ETRAMP antisense transcript levels dropped during parasite invasion, while sense transcript levels did not. Expression is plotted in units of log2(FPKM + 1). (E) Multi-exonic lncRNAs are encoded in the PfGDV1 region on chromosome nine, antisense to PfGDV1 and between PfGDV1 and GEXP22. Refer to (A)/(B) for a description of tracks. (F)/(G) Pearson correlation between the PfGDV1 sense-antisense pair was 0.96, while Pearson correlation between the divergent intergenic lncRNA and GEXP22 pair was 0.46 during the 56-hour time course. Expression is plotted in units of log2(FPKM + 1). (H) As we have previously described, the telomere-associated repetitive element (TARE) 2–3 region transcribes a family of lncRNA-TAREs, with transcription always proceeding towards the telomere [41]. For example, lncRNA-TARE-2 L is transcribed on the left arm of chromosome two. Pf3D7_0200100 is a subtelomeric upsB-type PfEMP1-encoding var gene. Boundaries of the telomere, TAREs 1–5, and Rep20 are shown in purple. See (A)/(B) for a further description of tracks. (I) Plotting the expression level of 22 lncRNA-TARE family members showed that lncRNA-TARE expression was co-regulated, with maximal firing coinciding with parasite invasion. Expression is plotted in units of log2(FPKM + 1). (J) Pearson correlation between lncRNA-TARE-2 L and the neighboring PfEMP1-encoding var gene was −0.09 during the 56-hour time course. Expression is plotted in units of log2(FPKM + 1)
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Related In: Results  -  Collection

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Fig4: Notable lncRNAs include multi-exonic and telomere-associated transcripts. (A)/(B) Multi-exonic antisense transcripts span an apicoplast RNA methyltransferase precursor [PlasmoDB:Pf3D7_0218300] and an ETRAMP [PlasmoDB:Pf3D7_0936100] gene, respectively. Annotated gene models are shown in dark green and dark blue, and assembled transcript models are shown in light green and light blue. Reads from each 56-hour time course sample mapping to the (−) strand are shown below each horizontal axis in light green, while reads mapping to the (+) strand are shown above each horizontal axis in light blue. Intron reads are shown in purple. Uniqueness of 100mers is plotted in red as a mappability track. (C)/(D) Pearson correlation between the Pf3D7_0218300 sense-antisense pair and ETRAMP sense-antisense pair during the 56-hour time course was 0.20 and −0.50, respectively. Notably, Pf3D7_0218300 and ETRAMP antisense transcript levels dropped during parasite invasion, while sense transcript levels did not. Expression is plotted in units of log2(FPKM + 1). (E) Multi-exonic lncRNAs are encoded in the PfGDV1 region on chromosome nine, antisense to PfGDV1 and between PfGDV1 and GEXP22. Refer to (A)/(B) for a description of tracks. (F)/(G) Pearson correlation between the PfGDV1 sense-antisense pair was 0.96, while Pearson correlation between the divergent intergenic lncRNA and GEXP22 pair was 0.46 during the 56-hour time course. Expression is plotted in units of log2(FPKM + 1). (H) As we have previously described, the telomere-associated repetitive element (TARE) 2–3 region transcribes a family of lncRNA-TAREs, with transcription always proceeding towards the telomere [41]. For example, lncRNA-TARE-2 L is transcribed on the left arm of chromosome two. Pf3D7_0200100 is a subtelomeric upsB-type PfEMP1-encoding var gene. Boundaries of the telomere, TAREs 1–5, and Rep20 are shown in purple. See (A)/(B) for a further description of tracks. (I) Plotting the expression level of 22 lncRNA-TARE family members showed that lncRNA-TARE expression was co-regulated, with maximal firing coinciding with parasite invasion. Expression is plotted in units of log2(FPKM + 1). (J) Pearson correlation between lncRNA-TARE-2 L and the neighboring PfEMP1-encoding var gene was −0.09 during the 56-hour time course. Expression is plotted in units of log2(FPKM + 1)
Mentions: Based on the diverse characteristics examined above, we searched for transcripts with exceptional properties. For example, we found that a putative Apicoplast RNA methyltransferase precursor [PlasmoDB:Pf3D7_0218300] and an Early Transcribed Membrane Protein [ETRAMP; PlasmoDB:Pf3D7_0936100] transcribe multi-exonic antisense RNAs across their full gene bodies [Fig. 4A and B]. Expression of the Apicoplast RNA methyltransferase precursor sense-antisense pair was not particularly correlated (ρ = .20), while expression of the ETRAMP sense-antisense pair was moderately anti-correlated (ρ = −.50) [Fig. 4C and D]. Interestingly, ETRAMP antisense transcription was substantially higher than ETRAMP sense transcription, reaching a maximum FPKM of 550 in early stages. This was the highest expression level observed for predicted P. falciparum antisense RNAs at any stage. Both the Apicoplast RNA methyltransferase precursor and ETRAMP antisense RNAs also demonstrated the 48 hpi expression drop phenomenon, though their sense partners did not exhibit this pattern.Fig. 4

Bottom Line: This approach enabled the annotation of over one thousand lncRNA transcript models and their comprehensive global analysis: coding prediction, periodicity, stage-specificity, correlation, GC content, length, location relative to annotated transcripts, and splicing.As compared to neighboring mRNAs, the expression of antisense-sense pairs was significantly anti-correlated during blood stage development, indicating transcriptional interference.We also validated that P. falciparum produces circRNAs, which is notable given the lack of RNA interference in the organism, and discovered that a highly expressed, five-exon antisense RNA is poised to regulate P. falciparum gametocyte development 1 (PfGDV1), a gene required for early sexual commitment events.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA. k8broadbent@post.harvard.edu.

ABSTRACT

Background: The human malaria parasite Plasmodium falciparum has a complex and multi-stage life cycle that requires extensive and precise gene regulation to allow invasion and hijacking of host cells, transmission, and immune escape. To date, the regulatory elements orchestrating these critical parasite processes remain largely unknown. Yet it is becoming increasingly clear that long non-coding RNAs (lncRNAs) could represent a missing regulatory layer across a broad range of organisms.

Results: To investigate the regulatory capacity of lncRNA in P. falciparum, we harvested fifteen samples from two time-courses. Our sample set profiled 56 h of P. falciparum blood stage development. We then developed and validated strand-specific, non-polyA-selected RNA sequencing methods, and pursued the first assembly of P. falciparum strand-specific transcript structures from RNA sequencing data. This approach enabled the annotation of over one thousand lncRNA transcript models and their comprehensive global analysis: coding prediction, periodicity, stage-specificity, correlation, GC content, length, location relative to annotated transcripts, and splicing. We validated the complete splicing structure of three lncRNAs with compelling properties. Non-polyA-selected deep sequencing also enabled the prediction of hundreds of intriguing P. falciparum circular RNAs, six of which we validated experimentally.

Conclusions: We found that a subset of lncRNAs, including all subtelomeric lncRNAs, strongly peaked in expression during invasion. By contrast, antisense transcript levels significantly dropped during invasion. As compared to neighboring mRNAs, the expression of antisense-sense pairs was significantly anti-correlated during blood stage development, indicating transcriptional interference. We also validated that P. falciparum produces circRNAs, which is notable given the lack of RNA interference in the organism, and discovered that a highly expressed, five-exon antisense RNA is poised to regulate P. falciparum gametocyte development 1 (PfGDV1), a gene required for early sexual commitment events.

No MeSH data available.


Related in: MedlinePlus