Inhibition of Cpeb3 ribozyme elevates CPEB3 protein expression and polyadenylation of its target mRNAs and enhances object location memory
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In this manuscript the authors describe the expression and regulatory function of a self-cleaving ribozyme in the Cpeb3 gene. This is an important study because although many self-cleaving ribozymes have been identified in the genome, the functions of these RNA enzymes even for molecular control of their target genes is mostly unknown. The manuscript provides solid data for the molecular function of the ribosome in gene regulation and its role in hippocampal learning. The study will be of interest to neurobiologists who study gene regulatory mechanisms in learning.
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Abstract
A self-cleaving ribozyme that maps to an intron of the cytoplasmic polyadenylation element-binding protein 3 ( Cpeb3 ) gene is thought to play a role in human episodic memory, but the underlying mechanisms mediating this effect are not known. We tested the activity of the murine sequence and found that the ribozyme’s self-scission half-life matches the time it takes an RNA polymerase to reach the immediate downstream exon, suggesting that the ribozyme-dependent intron cleavage is tuned to co-transcriptional splicing of the Cpeb3 mRNA. Our studies also reveal that the murine ribozyme modulates maturation of its harboring mRNA in both cultured cortical neurons and the hippocampus: inhibition of the ribozyme using an antisense oligonucleotide leads to increased CPEB3 protein expression, which enhances polyadenylation and translation of localized plasticity-related target mRNAs, and subsequently strengthens hippocampal-dependent long-term memory. These findings reveal a previously unknown role for self-cleaving ribozyme activity in regulating experience-induced co-transcriptional and local translational processes required for learning and memory.
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Author Response
Reviewer #3 (Public Review):
This manuscript uses ASO to inhibit the self-cleaving ribozyme within CPEB intron 3 and test its effect on CPEB3 expression and memory consolidation. The authors conclude that the intronic ribozyme negatively affects CPEB3 mRNA splicing and expression, and suggests its implications for experience-induced gene expression underlying learning and memory.
The strength of the manuscript is in its exploration of a potentially novel mechanism of regulating CPEB3 expression in learning and memory, a combination of both biochemical and behavioral approaches to gain a wide perspective of this regulatory mechanism, and the application of ASO in this context. The introduction is sufficiently detailed. Statistics are thorough and appropriate. If the results could be more robust, the mechanism would …
Author Response
Reviewer #3 (Public Review):
This manuscript uses ASO to inhibit the self-cleaving ribozyme within CPEB intron 3 and test its effect on CPEB3 expression and memory consolidation. The authors conclude that the intronic ribozyme negatively affects CPEB3 mRNA splicing and expression, and suggests its implications for experience-induced gene expression underlying learning and memory.
The strength of the manuscript is in its exploration of a potentially novel mechanism of regulating CPEB3 expression in learning and memory, a combination of both biochemical and behavioral approaches to gain a wide perspective of this regulatory mechanism, and the application of ASO in this context. The introduction is sufficiently detailed. Statistics are thorough and appropriate. If the results could be more robust, the mechanism would provide a novel target and venue to modify learning and memory paradigm.
The weakness of the manuscript is that the magnitude of the activity-dependent regulation of ribozyme, the effects of ASOs on CPEB3 expression (mRNA and protein) and downstream target gene expression, in vitro and in vivo, are generally weak, raising concerns about the robustness of the result. This may have caused some of the inconsistencies between the data presentation (see below). Also unclear is whether the ribozyme activity is physiologically regulated by experience without ASO interference.
While the statistics tests support corresponding figure panels and their conclusions. The manuscript can be significantly strengthened by additional evidence, clarification of some methodologies, and reconciling some inconsistent results.
The premise of a comparable timescale between transcription and ribozyme activity as the foundation of the whole thesis was based on in vitro measurement of self-scission half-life and a broadly generalized transcription rate (which actually varies significantly between genes). This premise is weak and needs direct experimental support.
The physiological relevance of the proposed mechanism has yet to be demonstrated without ASO interference.
Fig2b: how were total and uncleaved Ribozymes measured by qRT-PCR? Where are the primers' locations? If the two products were amplified using different primers, their subtraction to derive % cleavage would not be appropriate.
We thank the reviewer for the thoughtful review. We measured the levels of the total ribozyme by measuring a 220-bp amplicon that starts 18 nts downstream from the ribozyme cleavage site. The uncleaved ribozyme levels were measured using oligos that amplify a region of the intron that starts 45 nts upstream and ends 238 nts downstream of the ribozyme cleavage site. We added this information to the Table of primers in the manuscript. For all PCR oligos we established independent standard curves and calculated RNA levels independently of other amplicons, as noted in the Methods section and now specified in the Results section as well (Page 15). The measurements were thus appropriate for the calculation of the cleaved ribozyme fractions in the various experiments. The fraction ribozyme cleaved was calculated from the uncleaved fraction as the difference between uncleaved fraction and unity (1 – fraction uncleaved), now specified on page 16 of the manuscript. Fraction uncleaved was calculated as [uncleaved ribozyme]/[total ribozyme], as was done previously (see Salehi-Ashtiani et al. Science 313:1788-1792 or Webb et al. Science 326:953).
Line 400-403: shouldn't ribozyme-blocking ASO prevent ribozyme self-cleavage, and as a result should further increase ribozyme levels? This would contradict the result in fig3a.
We showed that the ribozyme is inhibited in vitro (Fig. 1F and 1G) and all our data are consistent with ASO inhibition of the ribozyme in cellulo and in vivo. However, we do not have direct evidence for this ribozyme inhibition in vivo, because such an experiment would require a single-molecule FRET-type sensitivity in cells and this assay has not been developed for ribozyme cleavage in cellulo or in vivo. We measured the ribozyme levels by RT-qPCR and observed lower ribozyme levels in presence of ASO in cultured neurons (Fig. 3A) as well as in vivo (Fig. 5B), which is nominally in contrast to the observations in vitro. However, in these situations we do not measure the co-transcriptional fate of the intron or the ribozyme; rather, we measure the levels of the intron after splicing (evidenced by the increased levels of spliced exons 2–3) when the intron is likely already being degraded. We also do not know what effect the ribozyme ASO has on the intron stability once splicing occurs. Understandably, this is a weakness of the study—and we are fully open about this result— however, given the abundance of evidence that the ribozyme ASO leads to increase of CPEB3 mRNA under all conditions tested, we feel that there is strong, if indirect, evidence that our model for the ribozyme function is correct. Future studies will examine this issue closer, but a definitive experimental investigation for the mechanism and timing of ribozyme inhibition and intron degradation is out of scope of this study.
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eLife assessment
In this manuscript the authors describe the expression and regulatory function of a self-cleaving ribozyme in the Cpeb3 gene. This is an important study because although many self-cleaving ribozymes have been identified in the genome, the functions of these RNA enzymes even for molecular control of their target genes is mostly unknown. The manuscript provides solid data for the molecular function of the ribosome in gene regulation and its role in hippocampal learning. The study will be of interest to neurobiologists who study gene regulatory mechanisms in learning.
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Reviewer #1 (Public Review):
In this manuscript the authors describe the expression and regulatory function of a self-cleaving ribozyme in the Cpeb3 gene. Cpeb3 knockout is associated with altered memory formation, and there are tempting correlations from the mid-2000s between a human CPEB3 SNP at the ribozyme cleavage site and memory performance, suggesting that regulation of Cpeb3 protein expression could impact memory. Here the authors test the impact of inhibiting Cpeb3 ribozyme self-excision with the hypothesis that this will promote splicing and Cpeb3 protein expression. They study the temporal regulation of ribozyme cleavage and find that it is in sync with transcription. Then they use their in vitro cleavage assay to identify an ASO that blocks cleavage. The validation of the effects of the ASO on ribozyme cleavage, and Cpeb3 …
Reviewer #1 (Public Review):
In this manuscript the authors describe the expression and regulatory function of a self-cleaving ribozyme in the Cpeb3 gene. Cpeb3 knockout is associated with altered memory formation, and there are tempting correlations from the mid-2000s between a human CPEB3 SNP at the ribozyme cleavage site and memory performance, suggesting that regulation of Cpeb3 protein expression could impact memory. Here the authors test the impact of inhibiting Cpeb3 ribozyme self-excision with the hypothesis that this will promote splicing and Cpeb3 protein expression. They study the temporal regulation of ribozyme cleavage and find that it is in sync with transcription. Then they use their in vitro cleavage assay to identify an ASO that blocks cleavage. The validation of the effects of the ASO on ribozyme cleavage, and Cpeb3 mRNA expression and processing in membrane depolarized neurons and in the hippocampus in vivo are rigorous and establish the molecular function of the ribozyme. The authors also show an increase in CPEB3 protein expression and increased expression (and polyadenylation) of known translational targets of CPEB3 in cultures and in vivo with the latter only in the presence of elevated neural activity, consistent with an effect on protein synthesis. The final part of the study assesses the regulation and function of CPEB3 in the context of learning and memory.
The significance of this study lies in the molecular analysis of the ribozyme function. This ribozyme is well established and the gene in which it lies has important links to synaptic plasticity. Gene regulation is known to be important in the context of learning and memory and this is a new mechanism that the authors show has the potential to influence this process.
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Reviewer #2 (Public Review):
For about four decades it has been known that RNA molecules can increase the rate of chemical reactions, just like the much more prevalent protein enzymes. Some have suggested that RNA enzymes, also called "ribozymes" were very important at the beginning of life, but that the importance was mostly erased when ribosomal protein synthesis emerged through evolution. The ribosome and spliceosome are two important examples of modern biological functions known to be catalyzed by RNA. In addition to these large RNA machines, the genomes of humans, and all domains of life, also contain a class of small ribozymes that catalyze self-cleavage of the RNA backbone. However, unlike RNA cleaving proteins that are well studied, there exists little evidence that the self-cleaving of RNA by ribozymes has important downstream …
Reviewer #2 (Public Review):
For about four decades it has been known that RNA molecules can increase the rate of chemical reactions, just like the much more prevalent protein enzymes. Some have suggested that RNA enzymes, also called "ribozymes" were very important at the beginning of life, but that the importance was mostly erased when ribosomal protein synthesis emerged through evolution. The ribosome and spliceosome are two important examples of modern biological functions known to be catalyzed by RNA. In addition to these large RNA machines, the genomes of humans, and all domains of life, also contain a class of small ribozymes that catalyze self-cleavage of the RNA backbone. However, unlike RNA cleaving proteins that are well studied, there exists little evidence that the self-cleaving of RNA by ribozymes has important downstream consequences. This new paper provides evidence that a ribozyme found in all mammals has an important role in memory formation. The authors found a way to block the ribozyme activity and then observe the effect on memory formation in mice, and in the expression of genes in neurons that are known to underly this memory formation process. The authors found that blocking the ribozyme activity in mouse brains actually improved their performance in a memory task. In addition, they found that blocking the ribozyme changed the expression of the gene in which the ribozyme is found (a gene called CPEB3), suggesting that the way the ribozyme effects memory is through controlling the expression of the gene where it is found. The paper confirms the biological importance of this ribozyme, and encourages further investigation into self-cleaving ribozymes in general. Interestingly, the ribozyme found in humans is in fact slower cleaving than most mammals, similar to the blocked ribozyme in these experiments, which brings up the intriguing possibility that the CPEB3 ribozyme is a part of what makes us human!
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Reviewer #3 (Public Review):
This manuscript uses ASO to inhibit the self-cleaving ribozyme within CPEB intron 3 and test its effect on CPEB3 expression and memory consolidation. The authors conclude that the intronic ribozyme negatively affects CPEB3 mRNA splicing and expression, and suggests its implications for experience-induced gene expression underlying learning and memory.
The strength of the manuscript is in its exploration of a potentially novel mechanism of regulating CPEB3 expression in learning and memory, a combination of both biochemical and behavioral approaches to gain a wide perspective of this regulatory mechanism, and the application of ASO in this context. The introduction is sufficiently detailed. Statistics are thorough and appropriate. If the results could be more robust, the mechanism would provide a novel target …Reviewer #3 (Public Review):
This manuscript uses ASO to inhibit the self-cleaving ribozyme within CPEB intron 3 and test its effect on CPEB3 expression and memory consolidation. The authors conclude that the intronic ribozyme negatively affects CPEB3 mRNA splicing and expression, and suggests its implications for experience-induced gene expression underlying learning and memory.
The strength of the manuscript is in its exploration of a potentially novel mechanism of regulating CPEB3 expression in learning and memory, a combination of both biochemical and behavioral approaches to gain a wide perspective of this regulatory mechanism, and the application of ASO in this context. The introduction is sufficiently detailed. Statistics are thorough and appropriate. If the results could be more robust, the mechanism would provide a novel target and venue to modify learning and memory paradigm.
The weakness of the manuscript is that the magnitude of the activity-dependent regulation of ribozyme, the effects of ASOs on CPEB3 expression (mRNA and protein) and downstream target gene expression, in vitro and in vivo, are generally weak, raising concerns about the robustness of the result. This may have caused some of the inconsistencies between the data presentation (see below). Also unclear is whether the ribozyme activity is physiologically regulated by experience without ASO interference.
While the statistics tests support corresponding figure panels and their conclusions. The manuscript can be significantly strengthened by additional evidence, clarification of some methodologies, and reconciling some inconsistent results.
The premise of a comparable timescale between transcription and ribozyme activity as the foundation of the whole thesis was based on in vitro measurement of self-scission half-life and a broadly generalized transcription rate (which actually varies significantly between genes). This premise is weak and needs direct experimental support.
The physiological relevance of the proposed mechanism has yet to be demonstrated without ASO interference.
Fig2b: how were total and uncleaved Ribozymes measured by qRT-PCR? Where are the primers' locations? If the two products were amplified using different primers, their subtraction to derive % cleavage would not be appropriate.
Line 400-403: shouldn't ribozyme-blocking ASO prevent ribozyme self-cleavage, and as a result should further increase ribozyme levels? This would contradict the result in fig3a. -
