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Synaptic vesicles contain small ribonucleic acids (sRNAs) including transfer RNA fragments (trfRNA) and microRNAs (miRNA).

Li H, Wu C, Aramayo R, Sachs MS, Harlow ML - Sci Rep (2015)

Bottom Line: In addition to classic neurotransmitters, we have found that synaptic vesicles isolated from the electric organ of Torpedo californica, a model cholinergic synapse, contain small ribonucleic acids (sRNAs), primarily the 5' ends of transfer RNAs (tRNAs) termed tRNA fragments (trfRNAs).This discovery suggests that, in addition to inducing changes in local dendritic excitability through the release of neurotransmitters, SVs may, through the release of specific trfRNAs and miRNAs, directly regulate local protein synthesis.We believe these findings have broad implications for the study of chemical synaptic transmission.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Texas A&M University, TAMU 3258, College Station, TX 77843-3474 USA.

ABSTRACT
Synaptic vesicles (SVs) are neuronal presynaptic organelles that load and release neurotransmitter at chemical synapses. In addition to classic neurotransmitters, we have found that synaptic vesicles isolated from the electric organ of Torpedo californica, a model cholinergic synapse, contain small ribonucleic acids (sRNAs), primarily the 5' ends of transfer RNAs (tRNAs) termed tRNA fragments (trfRNAs). To test the evolutionary conservation of SV sRNAs we examined isolated SVs from the mouse central nervous system (CNS). We found abundant levels of sRNAs in mouse SVs, including trfRNAs and micro RNAs (miRNAs) known to be involved in transcriptional and translational regulation. This discovery suggests that, in addition to inducing changes in local dendritic excitability through the release of neurotransmitters, SVs may, through the release of specific trfRNAs and miRNAs, directly regulate local protein synthesis. We believe these findings have broad implications for the study of chemical synaptic transmission.

No MeSH data available.


Related in: MedlinePlus

SVs isolated from the mouse CNS contain sRNAs.(a) Electron micrograph image of negatively stained vesicles isolated from the mouse CNS. Sample contains abundant ~40 nm vesicles (some marked with arrows). (b) Western-blot analysis of the synaptic vesicles during purification. The SV protein synaptophysin was used as a marker during isolation. The isolation procedure includes the collection of the orginal slurry (Sl), two centrifugation supernatants and pellets (S1, P1 and S2, P2), followed by a sucrose density gradient centrifugation and collection of the SV fluffy layer (FL). Further purification using size exclusion chromatography yields the final, isolated sample of SVs (SV). (c) Abundant sRNAs co-enrich with the synaptic vesicles (SV). These sRNAs are stable under high pH (pH10), or in the presence of detergent (DM). After addition of RNase much of the RNA is degraded; however an RNase resistant ~32 nt and lower ~20 nt band persists (RNase). The RNAse resistant band of RNA can be partially degraded in the presence of high pH, detergent, and RNAse (pHDR). (d) Northern analysis of RNA isolated from SVs verifies that the 5′-trfRNAGlu sequence, the second most abundant sequence in mouse CNS SV, was not a product of RNase treatment. RNA isolated from SVs purely isolated (SV), at pH10 (pH10), in detergent (DM), treated with RNase (RNase), and treated simultaneously with pH10, detergent, and RNase (pHDR). In the mouse CNS, both full-length tRNAGluCUC and 5′-trfRNAGlu co-enrich with the SVs, but only 5′-trfRNAGlu is resistant to RNase treatment. (e) The most abundant species of sRNA in mouse was a 5′ fragment of a Y RNA: Ro-associated Y1 (RNY1):5′-fRNY1. Like the 5′-trfRNAGlu, full length and precursor sequences of RNY1 co-enrich with the SVs. (c–e) Bands of gel underlined (_) indicate a 20-fold reduction of sample loaded. (f) The miRNA and RISC associated protein AGO2 co-enriches with SVs, however it is susceptible to trypsin degradation (0–20 minute exposure) indicating it does not reside within the lumen of the SVs. The SV protein synaptophysin, which has trypsin cleavage domains residing within the lumen of the SVs, is not degraded.
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f4: SVs isolated from the mouse CNS contain sRNAs.(a) Electron micrograph image of negatively stained vesicles isolated from the mouse CNS. Sample contains abundant ~40 nm vesicles (some marked with arrows). (b) Western-blot analysis of the synaptic vesicles during purification. The SV protein synaptophysin was used as a marker during isolation. The isolation procedure includes the collection of the orginal slurry (Sl), two centrifugation supernatants and pellets (S1, P1 and S2, P2), followed by a sucrose density gradient centrifugation and collection of the SV fluffy layer (FL). Further purification using size exclusion chromatography yields the final, isolated sample of SVs (SV). (c) Abundant sRNAs co-enrich with the synaptic vesicles (SV). These sRNAs are stable under high pH (pH10), or in the presence of detergent (DM). After addition of RNase much of the RNA is degraded; however an RNase resistant ~32 nt and lower ~20 nt band persists (RNase). The RNAse resistant band of RNA can be partially degraded in the presence of high pH, detergent, and RNAse (pHDR). (d) Northern analysis of RNA isolated from SVs verifies that the 5′-trfRNAGlu sequence, the second most abundant sequence in mouse CNS SV, was not a product of RNase treatment. RNA isolated from SVs purely isolated (SV), at pH10 (pH10), in detergent (DM), treated with RNase (RNase), and treated simultaneously with pH10, detergent, and RNase (pHDR). In the mouse CNS, both full-length tRNAGluCUC and 5′-trfRNAGlu co-enrich with the SVs, but only 5′-trfRNAGlu is resistant to RNase treatment. (e) The most abundant species of sRNA in mouse was a 5′ fragment of a Y RNA: Ro-associated Y1 (RNY1):5′-fRNY1. Like the 5′-trfRNAGlu, full length and precursor sequences of RNY1 co-enrich with the SVs. (c–e) Bands of gel underlined (_) indicate a 20-fold reduction of sample loaded. (f) The miRNA and RISC associated protein AGO2 co-enriches with SVs, however it is susceptible to trypsin degradation (0–20 minute exposure) indicating it does not reside within the lumen of the SVs. The SV protein synaptophysin, which has trypsin cleavage domains residing within the lumen of the SVs, is not degraded.

Mentions: We hypothesized that synaptic vesicles residing in the CNS might also contain sRNAs. To test this, we isolated SVs from the mouse brain of Swiss Webster mice. As expected, the mouse brain SVs were ~40 nm in diameter (Fig. 4a) and the synaptic vesicle marker synaptophysin co-purified with SVs during isolation (Fig. 4b; Supplemental Fig. 4). Once again, SVs were collected from the middle of the 0.6 M (1.07 g/ml density) sucrose gradient layer, well above the 1.2 M (1.17 g/ml) sucrose layer used to isolate exosomes1819 or detect exosome markers20. We treated the mouse SVs with TRIzol and found that the preparation was enriched in sRNAs (Fig. 4c). As with the T. californica SVs, we tested the mouse SV RNA under three conditions. We raised the buffer to pH10, but saw little difference. Next we added the detergent DM (also at pH10) and observed little difference between the RNA we isolated from the SV prep. When we added RNase to the SV prep we had a dramatic reduction (~38 fold; Supplemental Table 2) in the amount of total RNA extracted; however, two bands of resistant RNA at ~32 nt and ~21 nt remained. These bands appear to be partially degraded when we combined pH10, membrane detergent (DM), and RNase (Fig. 4c).


Synaptic vesicles contain small ribonucleic acids (sRNAs) including transfer RNA fragments (trfRNA) and microRNAs (miRNA).

Li H, Wu C, Aramayo R, Sachs MS, Harlow ML - Sci Rep (2015)

SVs isolated from the mouse CNS contain sRNAs.(a) Electron micrograph image of negatively stained vesicles isolated from the mouse CNS. Sample contains abundant ~40 nm vesicles (some marked with arrows). (b) Western-blot analysis of the synaptic vesicles during purification. The SV protein synaptophysin was used as a marker during isolation. The isolation procedure includes the collection of the orginal slurry (Sl), two centrifugation supernatants and pellets (S1, P1 and S2, P2), followed by a sucrose density gradient centrifugation and collection of the SV fluffy layer (FL). Further purification using size exclusion chromatography yields the final, isolated sample of SVs (SV). (c) Abundant sRNAs co-enrich with the synaptic vesicles (SV). These sRNAs are stable under high pH (pH10), or in the presence of detergent (DM). After addition of RNase much of the RNA is degraded; however an RNase resistant ~32 nt and lower ~20 nt band persists (RNase). The RNAse resistant band of RNA can be partially degraded in the presence of high pH, detergent, and RNAse (pHDR). (d) Northern analysis of RNA isolated from SVs verifies that the 5′-trfRNAGlu sequence, the second most abundant sequence in mouse CNS SV, was not a product of RNase treatment. RNA isolated from SVs purely isolated (SV), at pH10 (pH10), in detergent (DM), treated with RNase (RNase), and treated simultaneously with pH10, detergent, and RNase (pHDR). In the mouse CNS, both full-length tRNAGluCUC and 5′-trfRNAGlu co-enrich with the SVs, but only 5′-trfRNAGlu is resistant to RNase treatment. (e) The most abundant species of sRNA in mouse was a 5′ fragment of a Y RNA: Ro-associated Y1 (RNY1):5′-fRNY1. Like the 5′-trfRNAGlu, full length and precursor sequences of RNY1 co-enrich with the SVs. (c–e) Bands of gel underlined (_) indicate a 20-fold reduction of sample loaded. (f) The miRNA and RISC associated protein AGO2 co-enriches with SVs, however it is susceptible to trypsin degradation (0–20 minute exposure) indicating it does not reside within the lumen of the SVs. The SV protein synaptophysin, which has trypsin cleavage domains residing within the lumen of the SVs, is not degraded.
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Related In: Results  -  Collection

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f4: SVs isolated from the mouse CNS contain sRNAs.(a) Electron micrograph image of negatively stained vesicles isolated from the mouse CNS. Sample contains abundant ~40 nm vesicles (some marked with arrows). (b) Western-blot analysis of the synaptic vesicles during purification. The SV protein synaptophysin was used as a marker during isolation. The isolation procedure includes the collection of the orginal slurry (Sl), two centrifugation supernatants and pellets (S1, P1 and S2, P2), followed by a sucrose density gradient centrifugation and collection of the SV fluffy layer (FL). Further purification using size exclusion chromatography yields the final, isolated sample of SVs (SV). (c) Abundant sRNAs co-enrich with the synaptic vesicles (SV). These sRNAs are stable under high pH (pH10), or in the presence of detergent (DM). After addition of RNase much of the RNA is degraded; however an RNase resistant ~32 nt and lower ~20 nt band persists (RNase). The RNAse resistant band of RNA can be partially degraded in the presence of high pH, detergent, and RNAse (pHDR). (d) Northern analysis of RNA isolated from SVs verifies that the 5′-trfRNAGlu sequence, the second most abundant sequence in mouse CNS SV, was not a product of RNase treatment. RNA isolated from SVs purely isolated (SV), at pH10 (pH10), in detergent (DM), treated with RNase (RNase), and treated simultaneously with pH10, detergent, and RNase (pHDR). In the mouse CNS, both full-length tRNAGluCUC and 5′-trfRNAGlu co-enrich with the SVs, but only 5′-trfRNAGlu is resistant to RNase treatment. (e) The most abundant species of sRNA in mouse was a 5′ fragment of a Y RNA: Ro-associated Y1 (RNY1):5′-fRNY1. Like the 5′-trfRNAGlu, full length and precursor sequences of RNY1 co-enrich with the SVs. (c–e) Bands of gel underlined (_) indicate a 20-fold reduction of sample loaded. (f) The miRNA and RISC associated protein AGO2 co-enriches with SVs, however it is susceptible to trypsin degradation (0–20 minute exposure) indicating it does not reside within the lumen of the SVs. The SV protein synaptophysin, which has trypsin cleavage domains residing within the lumen of the SVs, is not degraded.
Mentions: We hypothesized that synaptic vesicles residing in the CNS might also contain sRNAs. To test this, we isolated SVs from the mouse brain of Swiss Webster mice. As expected, the mouse brain SVs were ~40 nm in diameter (Fig. 4a) and the synaptic vesicle marker synaptophysin co-purified with SVs during isolation (Fig. 4b; Supplemental Fig. 4). Once again, SVs were collected from the middle of the 0.6 M (1.07 g/ml density) sucrose gradient layer, well above the 1.2 M (1.17 g/ml) sucrose layer used to isolate exosomes1819 or detect exosome markers20. We treated the mouse SVs with TRIzol and found that the preparation was enriched in sRNAs (Fig. 4c). As with the T. californica SVs, we tested the mouse SV RNA under three conditions. We raised the buffer to pH10, but saw little difference. Next we added the detergent DM (also at pH10) and observed little difference between the RNA we isolated from the SV prep. When we added RNase to the SV prep we had a dramatic reduction (~38 fold; Supplemental Table 2) in the amount of total RNA extracted; however, two bands of resistant RNA at ~32 nt and ~21 nt remained. These bands appear to be partially degraded when we combined pH10, membrane detergent (DM), and RNase (Fig. 4c).

Bottom Line: In addition to classic neurotransmitters, we have found that synaptic vesicles isolated from the electric organ of Torpedo californica, a model cholinergic synapse, contain small ribonucleic acids (sRNAs), primarily the 5' ends of transfer RNAs (tRNAs) termed tRNA fragments (trfRNAs).This discovery suggests that, in addition to inducing changes in local dendritic excitability through the release of neurotransmitters, SVs may, through the release of specific trfRNAs and miRNAs, directly regulate local protein synthesis.We believe these findings have broad implications for the study of chemical synaptic transmission.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Texas A&M University, TAMU 3258, College Station, TX 77843-3474 USA.

ABSTRACT
Synaptic vesicles (SVs) are neuronal presynaptic organelles that load and release neurotransmitter at chemical synapses. In addition to classic neurotransmitters, we have found that synaptic vesicles isolated from the electric organ of Torpedo californica, a model cholinergic synapse, contain small ribonucleic acids (sRNAs), primarily the 5' ends of transfer RNAs (tRNAs) termed tRNA fragments (trfRNAs). To test the evolutionary conservation of SV sRNAs we examined isolated SVs from the mouse central nervous system (CNS). We found abundant levels of sRNAs in mouse SVs, including trfRNAs and micro RNAs (miRNAs) known to be involved in transcriptional and translational regulation. This discovery suggests that, in addition to inducing changes in local dendritic excitability through the release of neurotransmitters, SVs may, through the release of specific trfRNAs and miRNAs, directly regulate local protein synthesis. We believe these findings have broad implications for the study of chemical synaptic transmission.

No MeSH data available.


Related in: MedlinePlus