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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

Cholinergic SVs from the electric organ of Torpedo californica contain RNA.(a) Electron micrograph of negatively stained SVs isolated and enriched from electroplaque tissue. Sample contains abundant ~80 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 the enrichment. The isolation procedure includes the collection of the original 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, enriched sample of SVs (SV). Purified vesicles were tested by immunoblot and found to be positive for the ~60 kD vesicle acetylcholine transporter (VAChT). (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 band persists (RNase). The RNase resistant band of RNA can be degraded in the presence of high pH, detergent, and RNase (pHDR). Bands of gel underlined (_) indicate a 20-fold reduction of sample loaded. (d) TIRF microscope images of SVs reveals that single SVs contain RNA, as demonstrated by triple-labeling of the SVs with the styryl dye FM4–64, VAChT, and the RNA dye SYTO12. Five representative vesicles shown from a total of 307. Scale bar = 1 μm.
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f1: Cholinergic SVs from the electric organ of Torpedo californica contain RNA.(a) Electron micrograph of negatively stained SVs isolated and enriched from electroplaque tissue. Sample contains abundant ~80 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 the enrichment. The isolation procedure includes the collection of the original 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, enriched sample of SVs (SV). Purified vesicles were tested by immunoblot and found to be positive for the ~60 kD vesicle acetylcholine transporter (VAChT). (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 band persists (RNase). The RNase resistant band of RNA can be degraded in the presence of high pH, detergent, and RNase (pHDR). Bands of gel underlined (_) indicate a 20-fold reduction of sample loaded. (d) TIRF microscope images of SVs reveals that single SVs contain RNA, as demonstrated by triple-labeling of the SVs with the styryl dye FM4–64, VAChT, and the RNA dye SYTO12. Five representative vesicles shown from a total of 307. Scale bar = 1 μm.

Mentions: We isolated synaptic vesicles from the electric organ of the Pacific ray T. californica in order to provide an abundant, homogenous preparation of cholinergic SVs14. We chose a freeze grinding method of isolation that has been shown by others to retain more of the SV neurotransmitter content while offering a similar SV enrichment (~20 fold) as other isolation procedures1617. In addition, we wanted to isolate SVs residing within classic synaptosomal boutons as well as those present at less structured synaptic varicosities. 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 markers2021. The size of the vesicles we isolated averaged ~80 nm (Fig. 1a), larger than SVs within the vertebrate CNS (~40 nm)22 or SVs found at vertebrate neuromuscular junctions (~50 nm)2324, but normal for vesicles from this preparation25. As further verification that the isolated vesicles were neuronal in origin, we found by western blot analysis that the synaptic vesicle marker synaptophysin26 was enriched during isolation and that the vesicular acetylcholine transporter (VAChT)27 was present in the final preparation (Fig. 1b; Supplemental Fig. 1).


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)

Cholinergic SVs from the electric organ of Torpedo californica contain RNA.(a) Electron micrograph of negatively stained SVs isolated and enriched from electroplaque tissue. Sample contains abundant ~80 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 the enrichment. The isolation procedure includes the collection of the original 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, enriched sample of SVs (SV). Purified vesicles were tested by immunoblot and found to be positive for the ~60 kD vesicle acetylcholine transporter (VAChT). (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 band persists (RNase). The RNase resistant band of RNA can be degraded in the presence of high pH, detergent, and RNase (pHDR). Bands of gel underlined (_) indicate a 20-fold reduction of sample loaded. (d) TIRF microscope images of SVs reveals that single SVs contain RNA, as demonstrated by triple-labeling of the SVs with the styryl dye FM4–64, VAChT, and the RNA dye SYTO12. Five representative vesicles shown from a total of 307. Scale bar = 1 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4597359&req=5

f1: Cholinergic SVs from the electric organ of Torpedo californica contain RNA.(a) Electron micrograph of negatively stained SVs isolated and enriched from electroplaque tissue. Sample contains abundant ~80 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 the enrichment. The isolation procedure includes the collection of the original 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, enriched sample of SVs (SV). Purified vesicles were tested by immunoblot and found to be positive for the ~60 kD vesicle acetylcholine transporter (VAChT). (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 band persists (RNase). The RNase resistant band of RNA can be degraded in the presence of high pH, detergent, and RNase (pHDR). Bands of gel underlined (_) indicate a 20-fold reduction of sample loaded. (d) TIRF microscope images of SVs reveals that single SVs contain RNA, as demonstrated by triple-labeling of the SVs with the styryl dye FM4–64, VAChT, and the RNA dye SYTO12. Five representative vesicles shown from a total of 307. Scale bar = 1 μm.
Mentions: We isolated synaptic vesicles from the electric organ of the Pacific ray T. californica in order to provide an abundant, homogenous preparation of cholinergic SVs14. We chose a freeze grinding method of isolation that has been shown by others to retain more of the SV neurotransmitter content while offering a similar SV enrichment (~20 fold) as other isolation procedures1617. In addition, we wanted to isolate SVs residing within classic synaptosomal boutons as well as those present at less structured synaptic varicosities. 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 markers2021. The size of the vesicles we isolated averaged ~80 nm (Fig. 1a), larger than SVs within the vertebrate CNS (~40 nm)22 or SVs found at vertebrate neuromuscular junctions (~50 nm)2324, but normal for vesicles from this preparation25. As further verification that the isolated vesicles were neuronal in origin, we found by western blot analysis that the synaptic vesicle marker synaptophysin26 was enriched during isolation and that the vesicular acetylcholine transporter (VAChT)27 was present in the final preparation (Fig. 1b; Supplemental Fig. 1).

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