Multi‐omic profiling reveals an RNA processing rheostat that predisposes to prostate cancer

Abstract Prostate cancer is the most commonly diagnosed malignancy and the third leading cause of cancer deaths. GWAS have identified variants associated with prostate cancer susceptibility; however, mechanistic and functional validation of these mutations is lacking. We used CRISPR‐Cas9 genome editing to introduce a missense variant identified in the ELAC2 gene, which encodes a dually localised nuclear and mitochondrial RNA processing enzyme, into the mouse Elac2 gene as well as to generate a prostate‐specific knockout of Elac2. These mutations caused enlargement and inflammation of the prostate and nodule formation. The Elac2 variant or knockout mice on the background of the transgenic adenocarcinoma of the mouse prostate (TRAMP) model show that Elac2 mutation with a secondary genetic insult exacerbated the onset and progression of prostate cancer. Multiomic profiling revealed defects in energy metabolism that activated proinflammatory and tumorigenic pathways as a consequence of impaired noncoding RNA processing and reduced protein synthesis. Our physiologically relevant models show that the ELAC2 variant is a predisposing factor for prostate cancer and identify changes that underlie the pathogenesis of this cancer.

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Comments:
In the Abstract, it is written that "....the ELAC2 variant is a predisposing factor for prostate cancer and provides a detailed molecular mechanism and physiologically relevant models of this cancer". What detailed molecular mechanism is provided? In order to elucidate the molecular mechanism, relevant mutants of ELAC2 enzymes should be studied in their molecular context. Such as having a defined 3' end processing system of precursor tRNA and then examining the role of wild type versus mutant ELAC2 enzymes. Also, it is unclear in the case of prostate cancer if unprocessed precursor tRNAs with retained 3' trailers are oncogenic molecules. If not, then these immature precursor tRNAs exert their effect via different pathways, such as DNA damage and repair. The 3' tRNA endoribonuclease cleaves RNAs other than precursor tRNAs. Long noncoding RNAs having tRNA-like structures, like MALAT1 and MEN (for example, see PMID: 34001866, PMID: 19041754), are cleaved by the 3' tRNA endoribonuclease. Thus, these transcripts are also candidates for being involved in prostate cancer, as these long noncoding RNA were shown to be implicated in different malignancies. It will be constructive to tune down the conclusion that defects in 3' trailer cleavage of precursor tRNAs lead to prostate cancer.
Specific notes: In page 3, The following long sentence may be shortened or rephrased: Numerous genetic and genome wide-association studies (GWAS) in the last decade have implicated specific genes in the development of sporadic and, in particular, hereditary prostate cancer, including hereditary prostate cancer 1 gene (HPC1)/ Ribonuclease L (RNASEL) (1q24-25) and HPC2 or ElaC Homolog Protein 2 (ELAC2) (17p12), both of which are the most relevant candidate genes for prostate cancer predisposition. Figure 2C: If a longer exposure of ELAC2 band is available, that will be helpful to highlight the KO vs WT and A537T. Figure 7G legend: " (G) ELAC2 is a regulates the levels of...". Edit.

Referee #2 (Comments on Novelty/Model System for Author):
The authors looked into how ELAC2 (gene coding for a nuclear and mitochondrial tRNA endonuclease) mutations were functionally contributing to the risk of prostate cancer. They therefore developed four in vivo mouse models and thoroughly examined and interrogated their molecular and physiological characteristics. The authors provide with compelling functional and mechanistic evidence showing that an ELAC2 A537T variant predisposes to prostate cancer. They provide with comprehensive evidence to show that the nuclear and mitochondrial transcriptomes and proteomes, miRNAs and small RNAs correlate a direct link between reduced ELAC2 levels and activity to predisposition to prostate cancer. This variant alters the maturation and therefore the function of non-coding RNAs such as tRNAs, lncRNAs and miRNAs which in turn alter immunometabolism. Specifically, the authors demonstrate how ELAC2 mutations cause prostate enlargement and inflammation which leads to prostate cancer on a TRAMP background. Moreover, the demonstrate how the ELAC2 A537T variant leads to inflammation, cell proliferation and differentiation in the prostate a direct impact of impaired tRNA processing that as a result causes transcriptomewide changes ultimately promoting prostate tumorigenesis. Their analysis indicates that defects in aerobic metabolism drive prostate tumorigenesis via the PERK-mediated unfolded protein response. The authors further indicate that ELAC2 is a rheostat that regulates miRNA homeostasis and as a result the ELAC2 variant leads to miRNA imbalance and potentiation of tumorigenesis in the prostates of the A537T-TRAMP mice. Lastly the authors made a rather important and unexpected discovery. They show that ELAC2 A537T variant leads to prostate tumorigenesis by reducing, as of previously undescribed, ELAC2 interactions with SFPQ and NONO, proteins essential in multiple processes RNA-binding proteins, thus affecting RNA and energy metabolism.
This work is of great significance because it establishes new and physiologically relevant models of prostate tumorigenesis and cancer, and could be of considerable impact in drug screens and therapeutic development as well as to the continued investigation of prostate cancer biology. The work is of exceptional quality and no edits are recommended.

Point-by-point response to the reviewers' comments
Reviewer #1: This manuscript, entitled "Multi-omic profiling reveals an RNA processing rheostat that predisposes to prostate cancer" by Stentenbachby et al, describes interesting findings that reveal that human disease-relevant mutations of ELAC2, 3' tRNA endoribonuclease, are predisposing factors of prostate cancer in mouse models. The models included homozygous prostate-specific knockout ELAC2 gene and full-gene homozygous ELAC2 A537T variant (equivalent to the human A541T substitution), along with their respective homozygous control mice. CRISPR-Cas9 genome editing was applied to introduce the A541T variant in the Elac2 gene, and homologous recombination to generate a prostate-specific deletion of Elac2. These were bred onto transgenic adenocarcinoma of mouse prostate (TRAMP) model that carries the T antigens of SV40 under the control of probasin promoter.
To show the effect of these mutations on prostate cancer risk, these strains, having ELAC2 gene knockout and A537T variant, were examined for molecular and physiological characteristics. Through performing nuclear, mitochondrial transcriptome, proteome, small RNAs and miRNAs wide-screen studies, it was argued that reduced steady state levels and activity of ELAC2 predispose to prostate cancer by weakening the function of non-coding RNAs, such as tRNAs, long noncoding RNAs and microRNAs, which seem to alter immunometabolism. This manuscript provides general, but extensive and valuable information about KO of ELAC2 and its A537T variant, found in the human population, that predispose to prostate cancer. However, the underlying precise molecular mechanism for the direct role of ELAC2 in prostate cancer remains unresolved. Though, the authors mentioned in the abstract that the molecular mechanism is determined. In general, the application of multi-omic profiling does not delineate the exact molecular mechanism responsible for a molecular process. Elucidation of the molecular mechanism by which ELAC2 directly causes prostate cancer will need additional functional approaches, including comparative enzymatic assays of the wild type and disease-relevant mutants of ELAC2 in vitro and in vivo, combined with rescue genetic experiments of the mutants by the use of wild type ELAC2 gene both in mice and then in vitro. It will also be helpful to substitute, if feasible, the mouse ELAC2 gene with human wild type and disease-related mutant ELAC2 genes. Nevertheless, the data presented in the manuscript is sufficient to support the conclusion that knockout of the 3' tRNA endoribonuclease and expression of clinically-relevant mutants of this endoribonuclease, promote prostate cancer in model animals.
We thank this reviewer for their advice and support of this work and address their insightful comments below. We have already created four new models of prostate inflammation and tumorigenesis, characterised their phenotypes, and generated transcriptomic data showing accumulation of 3′ tRNA precursors in vivo, therefore generating additional models where the mouse gene is substituted with the human gene is beyond the scope of the current study and would take several additional years of work. The point mutation introduced into the mouse ELAC2 gene alters an amino acid that is conserved in human ELAC2 and predisposes to prostate cancer, hence why we carried out this, more physiologically relevant, knock in, so that we do not introduce additional variables and potential 22nd Mar 2023 1st Authors' Response to Reviewers 2 incompatibilities that would occur if the human equivalent of the whole gene was introduced in the background of the mouse genome. This alternative approach is a nice suggestion from this reviewer, and hybrid data like this would complement our study as an interesting project for the future.
The in vitro cleavage activity of recombinantly produced ELAC2 bearing the A541T variant has previously been analysed (PMID: 12711671) and no significant change was observed compared to wild-type ELAC2, when equivalent amounts of protein were tested. In our work we show that the A541T variant reduces the stability of ELAC2 by ~30%, which results in decreased in vivo cleavage of ELAC2 target RNAs in the prostates of our different mouse models. Reduction in RNA processing as a consequence of the A541T variant explains its presence in the human population without the dramatic consequences of disease mutations that cause loss or severe reduction of ELAC2 activity resulting in early-onset cardiomyopathies (PMID: 23849775). Nevertheless, the long-term consequences of the changes induced by the A541T variant in combination with a second oncogenic insult manifest in altered cell signalling, inflammation and predisposition to prostate cancer.

Comments:
In the Abstract, it is written that "....the ELAC2 variant is a predisposing factor for prostate cancer and provides a detailed molecular mechanism and physiologically relevant models of this cancer". What detailed molecular mechanism is provided? In order to elucidate the molecular mechanism, relevant mutants of ELAC2 enzymes should be studied in their molecular context. Such as having a defined 3' end processing system of precursor tRNA and then examining the role of wild type versus mutant ELAC2 enzymes. Also, it is unclear in the case of prostate cancer if unprocessed precursor tRNAs with retained 3' trailers are oncogenic molecules. If not, then these immature precursor tRNAs exert their effect via different pathways, such as DNA damage and repair.
The 3' tRNA endoribonuclease cleaves RNAs other than precursor tRNAs. Long noncoding RNAs having tRNA-like structures, like MALAT1 and MEN (for example, see PMID: 34001866, PMID: 19041754), are cleaved by the 3' tRNA endoribonuclease. Thus, these transcripts are also candidates for being involved in prostate cancer, as these long noncoding RNA were shown to be implicated in different malignancies.
It will be constructive to tune down the conclusion that defects in 3' trailer cleavage of precursor tRNAs lead to prostate cancer.
We provide the molecular changes that lead to prostate tumorigenesis in the four different models relative to their two controls in the manuscript, and therefore we have clarified this in the revised manuscript as suggested by this reviewer. ELAC2 is not required for processing of the MALAT1 lncRNA, this is achieved by RNase P at it's 3′ end (PMID: 34253686 and PMID: 19041754), consistent with our RNA sequencing findings. We did not mean to imply that the accumulation of 3' precursors is causing the tumorigenicity, rather that the lack of mature transcripts as a consequence of impaired RNA processing exerts effects on the pathways that are changing. We have edited the relevant text to clarify this distinction. In addition, we have edited this conclusion as suggested by the reviewer in the abstract and the concluding sentence in the introduction.
3 Specific notes: In page 3, The following long sentence may be shortened or rephrased: Numerous genetic and genome wide-association studies (GWAS) in the last decade have implicated specific genes in the development of sporadic and, in particular, hereditary prostate cancer, including hereditary prostate cancer 1 gene (HPC1)/ Ribonuclease L (RNASEL) (1q24-25) and HPC2 or ElaC Homolog Protein 2 (ELAC2) (17p12), both of which are the most relevant candidate genes for prostate cancer predisposition.
We have shortened this sentence as suggested by breaking it into two sentences. Figure 2C: If a longer exposure of ELAC2 band is available, that will be helpful to highlight the KO vs WT and A537T.
The blot included in this figure is the longest exposure, although there is some background, this is the only antibody on the market that can detect ELAC2 in mouse tissues. Figure 7G legend: " (G) ELAC2 is a regulates the levels of...". Edit.
Thank you for spotting this typographical error, we have corrected this.
Reviewer #2 (Remarks to the Author): The authors looked into how ELAC2 (gene coding for a nuclear and mitochondrial tRNA endonuclease) mutations were functionally contributing to the risk of prostate cancer. They therefore developed four in vivo mouse models and thoroughly examined and interrogated their molecular and physiological characteristics.
The authors provide with compelling functional and mechanistic evidence showing that an ELAC2 A537T variant predisposes to prostate cancer. They provide with comprehensive evidence to show that the nuclear and mitochondrial transcriptomes and proteomes, miRNAs and small RNAs correlate a direct link between reduced ELAC2 levels and activity to predisposition to prostate cancer. This variant alters the maturation and therefore the function of non-coding RNAs such as tRNAs, lncRNAs and miRNAs which in turn alter immunometabolism.
Specifically, the authors demonstrate how ELAC2 mutations cause prostate enlargement and inflammation which leads to prostate cancer on a TRAMP background. Moreover, the demonstrate how the ELAC2 A537T variant leads to inflammation, cell proliferation and differentiation in the prostate a direct impact of impaired tRNA processing that as a result causes transcriptome-wide changes ultimately promoting prostate tumorigenesis. Their analysis indicates that defects in aerobic metabolism drive prostate tumorigenesis via the PERK-mediated unfolded protein response.

4
The authors further indicate that ELAC2 is a rheostat that regulates miRNA homeostasis and as a result the ELAC2 variant leads to miRNA imbalance and potentiation of tumorigenesis in the prostates of the A537T-TRAMP mice.
Lastly the authors made a rather important and unexpected discovery. They show that ELAC2 A537T variant leads to prostate tumorigenesis by reducing, as of previously undescribed, ELAC2 interactions with SFPQ and NONO, proteins essential in multiple processes RNA-binding proteins, thus affecting RNA and energy metabolism.
This work is of great significance because it establishes new and physiologically relevant models of prostate tumorigenesis and cancer, and could be of considerable impact in drug screens and therapeutic development as well as to the continued investigation of prostate cancer biology. The work is of exceptional quality and no edits are recommended.
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Lise Roth
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