Limits...
Small RNAs from plants, bacteria and fungi within the order Hypocreales are ubiquitous in human plasma.

Beatty M, Guduric-Fuchs J, Brown E, Bridgett S, Chakravarthy U, Hogg RE, Simpson DA - BMC Genomics (2014)

Bottom Line: The human microbiome plays a significant role in maintaining normal physiology.The source and functions of these molecules remain to be determined, but the specific profiles are likely to reflect health status.The potential to provide biomarkers of diet and for the diagnosis and prognosis of human disease is immense.

View Article: PubMed Central - PubMed

Affiliation: Centre for Experimental Medicine, Queen's University Belfast, Belfast, Northern Ireland, UK. David.Simpson@qub.ac.uk.

ABSTRACT

Background: The human microbiome plays a significant role in maintaining normal physiology. Changes in its composition have been associated with bowel disease, metabolic disorders and atherosclerosis. Sequences of microbial origin have been observed within small RNA sequencing data obtained from blood samples. The aim of this study was to characterise the microbiome from which these sequences are derived.

Results: Abundant non-human small RNA sequences were identified in plasma and plasma exosomal samples. Assembly of these short sequences into longer contigs was the pivotal novel step in ascertaining their origin by BLAST searches. Most reads mapped to rRNA sequences. The taxonomic profiles of the microbes detected were very consistent between individuals but distinct from microbiomes reported at other sites. The majority of bacterial reads were from the phylum Proteobacteria, whilst for 5 of 6 individuals over 90% of the more abundant fungal reads were from the phylum Ascomycota; of these over 90% were from the order Hypocreales. Many contigs were from plants, presumably of dietary origin. In addition, extremely abundant small RNAs derived from human Y RNAs were detected.

Conclusions: A characteristic profile of a subset of the human microbiome can be obtained by sequencing small RNAs present in the blood. The source and functions of these molecules remain to be determined, but the specific profiles are likely to reflect health status. The potential to provide biomarkers of diet and for the diagnosis and prognosis of human disease is immense.

Show MeSH

Related in: MedlinePlus

Small RNAs derived from the non-coding hY4 RNA present in plasma. (A) The predicted secondary structure of hY4 is shown in dot-bracket notation above the sequence and the reads mapping to the 5p and 3p arms indicated below (numbers refer to the reads detected in sample 1a). The positions of the most abundant 5p and 3p reads (and much less frequent short reads) are indicated by arrows adjacent to the hY4 structure. (B) Custom Taqman small RNA assays targeting the hY4-5p or 3p RNAs corresponding to the most abundant reads amplified products several threshold cycles before individual microRNAs (eg miR-22 in sample 1a). (C) RT-PCR with primers specific for the putative hY4 fragments and performed upon RNA that had been polyadenylated, amplified products with lengths consistent with the presence of the small RNA templates detected in the sequencing rather than full length hY4 RNA. A product of the predicted size (79 bp) was detected with the hY4 5p primer, whereas a longer product of 143 bp would have been amplified from full length hY4 RNA. M: Marker, sizes in bp; Lane 1, hY4 5p; Lane 2, No RT; Lane 3, hY4 3p; Lane 4, No RT.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4230795&req=5

Fig3: Small RNAs derived from the non-coding hY4 RNA present in plasma. (A) The predicted secondary structure of hY4 is shown in dot-bracket notation above the sequence and the reads mapping to the 5p and 3p arms indicated below (numbers refer to the reads detected in sample 1a). The positions of the most abundant 5p and 3p reads (and much less frequent short reads) are indicated by arrows adjacent to the hY4 structure. (B) Custom Taqman small RNA assays targeting the hY4-5p or 3p RNAs corresponding to the most abundant reads amplified products several threshold cycles before individual microRNAs (eg miR-22 in sample 1a). (C) RT-PCR with primers specific for the putative hY4 fragments and performed upon RNA that had been polyadenylated, amplified products with lengths consistent with the presence of the small RNA templates detected in the sequencing rather than full length hY4 RNA. A product of the predicted size (79 bp) was detected with the hY4 5p primer, whereas a longer product of 143 bp would have been amplified from full length hY4 RNA. M: Marker, sizes in bp; Lane 1, hY4 5p; Lane 2, No RT; Lane 3, hY4 3p; Lane 4, No RT.

Mentions: The proportions of reads annotated to human genes are illustrated in Figure 2A (absolute numbers in Additional file1). As expected, a large proportion of reads represented microRNAs, but remarkably, in the whole plasma samples prepared in this study, a similar proportion mapped to Y RNAs. Y RNAs are small cytoplasmic non-coding RNAs that can be cleaved to form smaller RNAs independently of the microRNA pathway[29]. The vast majority of reads (>99%) mapped to hY4, with small numbers to hY5, hY3 and hY1. A smaller but significant number of Y RNA sequences were present in the plasma exosome samples. In small RNA sequencing datasets from whole blood, which included cellular RNAs (GEO accession GSE46579), hy4-derived RNAs were present at levels comparable to an abundant microRNA[30]. The differences in Y RNA abundance observed between studies can be attributed to differences in sample collection (eg whole plasma or plasma exosomes) and library preparation, which result in differing distributions of small RNA read lengths (Additional file2: Figure S1). The small RNAs detected corresponded to the 5p and 3p arms of the predicted secondary structure of hY4 (Figure 3A). Taqman small RNA RT-qPCR assays employ a stem-loop reverse transcription primer and are therefore expected to be specific for the target small RNA and not detect the full length precursor RNA. Therefore the low Cp values observed with the assays targeting the most abundant hY4 sequences from each arm both confirmed the presence of these small RNAs in plasma and suggested that they are indeed much more abundant than any individual microRNA (Figure 3B). To further confirm the presence of hY4 fragments, RNA was polyadenylated, reverse transcribed with an oligo-dT adaptor and PCR performed with primers specific for the putative hY4 fragments. The size of the product amplified using the 5p primer was consistent with presence of the small RNA template detected in the sequencing rather than full length hY4 RNA (Figure 3C).Figure 2


Small RNAs from plants, bacteria and fungi within the order Hypocreales are ubiquitous in human plasma.

Beatty M, Guduric-Fuchs J, Brown E, Bridgett S, Chakravarthy U, Hogg RE, Simpson DA - BMC Genomics (2014)

Small RNAs derived from the non-coding hY4 RNA present in plasma. (A) The predicted secondary structure of hY4 is shown in dot-bracket notation above the sequence and the reads mapping to the 5p and 3p arms indicated below (numbers refer to the reads detected in sample 1a). The positions of the most abundant 5p and 3p reads (and much less frequent short reads) are indicated by arrows adjacent to the hY4 structure. (B) Custom Taqman small RNA assays targeting the hY4-5p or 3p RNAs corresponding to the most abundant reads amplified products several threshold cycles before individual microRNAs (eg miR-22 in sample 1a). (C) RT-PCR with primers specific for the putative hY4 fragments and performed upon RNA that had been polyadenylated, amplified products with lengths consistent with the presence of the small RNA templates detected in the sequencing rather than full length hY4 RNA. A product of the predicted size (79 bp) was detected with the hY4 5p primer, whereas a longer product of 143 bp would have been amplified from full length hY4 RNA. M: Marker, sizes in bp; Lane 1, hY4 5p; Lane 2, No RT; Lane 3, hY4 3p; Lane 4, No RT.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230795&req=5

Fig3: Small RNAs derived from the non-coding hY4 RNA present in plasma. (A) The predicted secondary structure of hY4 is shown in dot-bracket notation above the sequence and the reads mapping to the 5p and 3p arms indicated below (numbers refer to the reads detected in sample 1a). The positions of the most abundant 5p and 3p reads (and much less frequent short reads) are indicated by arrows adjacent to the hY4 structure. (B) Custom Taqman small RNA assays targeting the hY4-5p or 3p RNAs corresponding to the most abundant reads amplified products several threshold cycles before individual microRNAs (eg miR-22 in sample 1a). (C) RT-PCR with primers specific for the putative hY4 fragments and performed upon RNA that had been polyadenylated, amplified products with lengths consistent with the presence of the small RNA templates detected in the sequencing rather than full length hY4 RNA. A product of the predicted size (79 bp) was detected with the hY4 5p primer, whereas a longer product of 143 bp would have been amplified from full length hY4 RNA. M: Marker, sizes in bp; Lane 1, hY4 5p; Lane 2, No RT; Lane 3, hY4 3p; Lane 4, No RT.
Mentions: The proportions of reads annotated to human genes are illustrated in Figure 2A (absolute numbers in Additional file1). As expected, a large proportion of reads represented microRNAs, but remarkably, in the whole plasma samples prepared in this study, a similar proportion mapped to Y RNAs. Y RNAs are small cytoplasmic non-coding RNAs that can be cleaved to form smaller RNAs independently of the microRNA pathway[29]. The vast majority of reads (>99%) mapped to hY4, with small numbers to hY5, hY3 and hY1. A smaller but significant number of Y RNA sequences were present in the plasma exosome samples. In small RNA sequencing datasets from whole blood, which included cellular RNAs (GEO accession GSE46579), hy4-derived RNAs were present at levels comparable to an abundant microRNA[30]. The differences in Y RNA abundance observed between studies can be attributed to differences in sample collection (eg whole plasma or plasma exosomes) and library preparation, which result in differing distributions of small RNA read lengths (Additional file2: Figure S1). The small RNAs detected corresponded to the 5p and 3p arms of the predicted secondary structure of hY4 (Figure 3A). Taqman small RNA RT-qPCR assays employ a stem-loop reverse transcription primer and are therefore expected to be specific for the target small RNA and not detect the full length precursor RNA. Therefore the low Cp values observed with the assays targeting the most abundant hY4 sequences from each arm both confirmed the presence of these small RNAs in plasma and suggested that they are indeed much more abundant than any individual microRNA (Figure 3B). To further confirm the presence of hY4 fragments, RNA was polyadenylated, reverse transcribed with an oligo-dT adaptor and PCR performed with primers specific for the putative hY4 fragments. The size of the product amplified using the 5p primer was consistent with presence of the small RNA template detected in the sequencing rather than full length hY4 RNA (Figure 3C).Figure 2

Bottom Line: The human microbiome plays a significant role in maintaining normal physiology.The source and functions of these molecules remain to be determined, but the specific profiles are likely to reflect health status.The potential to provide biomarkers of diet and for the diagnosis and prognosis of human disease is immense.

View Article: PubMed Central - PubMed

Affiliation: Centre for Experimental Medicine, Queen's University Belfast, Belfast, Northern Ireland, UK. David.Simpson@qub.ac.uk.

ABSTRACT

Background: The human microbiome plays a significant role in maintaining normal physiology. Changes in its composition have been associated with bowel disease, metabolic disorders and atherosclerosis. Sequences of microbial origin have been observed within small RNA sequencing data obtained from blood samples. The aim of this study was to characterise the microbiome from which these sequences are derived.

Results: Abundant non-human small RNA sequences were identified in plasma and plasma exosomal samples. Assembly of these short sequences into longer contigs was the pivotal novel step in ascertaining their origin by BLAST searches. Most reads mapped to rRNA sequences. The taxonomic profiles of the microbes detected were very consistent between individuals but distinct from microbiomes reported at other sites. The majority of bacterial reads were from the phylum Proteobacteria, whilst for 5 of 6 individuals over 90% of the more abundant fungal reads were from the phylum Ascomycota; of these over 90% were from the order Hypocreales. Many contigs were from plants, presumably of dietary origin. In addition, extremely abundant small RNAs derived from human Y RNAs were detected.

Conclusions: A characteristic profile of a subset of the human microbiome can be obtained by sequencing small RNAs present in the blood. The source and functions of these molecules remain to be determined, but the specific profiles are likely to reflect health status. The potential to provide biomarkers of diet and for the diagnosis and prognosis of human disease is immense.

Show MeSH
Related in: MedlinePlus