Recombinant Xenopus laevis Methionine--tRNA ligase, mitochondrial (mars2), partial

What is mars2 and what is its function in Xenopus laevis?

Mars2 (Methionine--tRNA ligase, mitochondrial) is a gene that encodes a mitochondrial methionyl-tRNA synthetase, which is essential for the translation of mitochondrial genes . This enzyme specifically attaches methionine to its cognate tRNA, enabling protein synthesis within mitochondria. As mitochondria are responsible for cellular energy production through oxidative phosphorylation, mars2 plays a critical role in maintaining cellular respiration and ATP generation during the energy-demanding processes of embryonic development.

In Xenopus, researchers have identified both conventional mars2 transcripts and a unique mars2-like transcript with distinct expression patterns and structures . This diversity suggests specialized roles during different developmental stages.

How do conventional mars2 and mars2-like transcripts differ structurally?

The two distinct mars2-related transcripts in Xenopus exhibit significant structural differences:

This structural differentiation suggests that mars2-like is a chimeric transcript formed through the integration of retrotransposon elements, creating a unique variant with potentially distinct functions .

What are the expression patterns of mars2 during Xenopus embryonic development?

The conventional mars2 and mars2-like transcripts display remarkably different temporal expression profiles during Xenopus development:

Conventional mars2:

• Shows highest expression during the first three embryonic stages

• Gradually decreases until the blastula stage (stage 9)

• Is not affected by α-amanitin treatment, indicating it represents a maternal transcript present in the egg before fertilization

Mars2-like transcript:

• Displays low expression in early embryonic stages

• Begins to increase at blastula stage (stage 9) when conventional mars2 levels are decreasing

• Shows expression profile similar to the retrotransposon λ-olt 2-1

• Is significantly reduced by α-amanitin treatment, confirming it is newly transcribed by RNA polymerase II in embryos

The complementary expression patterns suggest that mars2-like may functionally substitute for conventional mars2 during later developmental stages, potentially representing a mechanism for developmental stage-specific regulation of mitochondrial translation .

What techniques can be used to detect and differentiate mars2 transcripts in Xenopus embryos?

Several molecular techniques can be employed to study mars2 variants in Xenopus:

• RT-qPCR with specific primers:

  • Design primers that target unique regions of each transcript

  • For mars2-like, primers spanning the junction between the retrotransposon LTR and mars2-homologous sequence are effective

  • Consider poly(A) tail status; oligo-dT primers for reverse transcription will only capture polyadenylated transcripts

• RNA-Seq analysis:

  • Transcriptome assembly mapped to the Xenopus laevis genome can identify mars2 transcript variants

  • Analysis of sequencing reads across splicing junctions can validate transcript structures

• PCR amplification and sequencing:

  • Primers located at the beginning of exon 1 and in exon 2 can amplify mars2-like transcripts

  • Sequencing of PCR products can validate splicing junctions

• α-amanitin treatment:

  • Injection of α-amanitin (RNA polymerase II inhibitor) differentially affects conventional mars2 (resistant) and mars2-like (sensitive)

  • This provides a functional way to distinguish maternal from zygotic transcripts

For visualization of expression patterns, rabies virus-based tools can be adapted for studying gene expression in Xenopus, as they allow for retrograde labeling and transgene expression, which could be applied to mars2 studies .

How can α-amanitin be used to distinguish between maternal and zygotic mars2 transcripts?

α-amanitin is a specific inhibitor of RNA polymerase II that serves as a powerful tool to differentiate between maternal transcripts (present in eggs before fertilization) and zygotic transcripts (newly synthesized after fertilization):

Experimental protocol:

• Inject α-amanitin at different concentrations into Xenopus embryos

• Collect embryos at specific developmental stages

• Extract RNA and perform RT-qPCR using transcript-specific primers

• Compare transcript levels between treated and control embryos

• Include controls such as pwp1 (maternal transcript) to validate results

This experimental approach provides valuable insights into the developmental regulation of mars2 variants and suggests distinct roles during embryonic development. The differential sensitivity to α-amanitin confirms that mars2-like is newly transcribed in embryos, while conventional mars2 is maternally provided .

What is the relationship between mars2-like transcript and retrotransposons?

The mars2-like transcript represents a fascinating example of retrotransposon influence on gene expression in Xenopus:

• Chimeric transcript structure:

  • The first exon and surrounding region shows 97% sequence identity to the LTR of the retrotransposon λ-olt 2-1

  • This suggests the LTR has been co-opted to serve as a promoter or regulatory element

• Developmental regulation:

  • The expression profile of mars2-like resembles that of λ-olt 2-1 itself

  • Both show increased expression at the blastula stage (stage 9)

• Transcriptional control:

  • Mars2-like is newly transcribed by RNA polymerase II in embryos, as evidenced by its sensitivity to α-amanitin

  • This differs from conventional mars2, which appears to be maternally loaded

• Functional implications:

  • The timing of mars2-like expression coincides with the decrease in conventional mars2, suggesting it may substitute for conventional mars2 function during later developmental stages

  • This represents a potential mechanism for developmental stage-specific regulation of mitochondrial translation

This relationship demonstrates how retrotransposon elements can be evolutionarily repurposed to create novel gene regulatory networks, potentially contributing to the unique developmental program of Xenopus.

How can CRISPR-Cas9 be used to study mars2 function in Xenopus development?

CRISPR-Cas9 genome editing provides powerful approaches for studying mars2 function in Xenopus:

• Knockout strategies:

  • Design guide RNAs targeting either common regions of both mars2 transcripts or specific regions unique to conventional mars2 or mars2-like

  • Inject Cas9 protein and gRNAs into fertilized eggs to generate F0 mosaic mutants

  • Analyze effects on mitochondrial function, energy metabolism, and developmental outcomes

• Truncation or domain-specific mutations:

  • Create specific mutations in functional domains to dissect the role of different parts of mars2

  • Target the unique first exon of mars2-like to specifically disrupt this transcript without affecting conventional mars2

• Knock-in approaches:

  • Insert reporter genes (e.g., GFP) in-frame with mars2 to track expression and localization

  • Create specific point mutations to model potential disease-associated variants

• Integration with viral tools:

  • Combine CRISPR-Cas9 with viral delivery systems such as rabies virus

  • This enables sophisticated spatial and temporal control of mars2 expression

When designing CRISPR experiments, include appropriate controls such as non-targeting gRNAs and rescue experiments with wild-type mRNA. Consider potential off-target effects and validate editing efficiency through sequencing.

How can rabies virus-based tools enhance mars2 research in Xenopus?

Recombinant rabies virus systems offer innovative approaches for studying mars2 in Xenopus:

• Cell-type specific labeling:

  • Glycoprotein-deleted rabies virus can retrogradely label neurons when injected into brain tissue

  • This approach can be adapted to identify cells expressing mars2 and analyze its distribution

• Transgene expression:

  • Rabies virus can express transgenes in Xenopus neurons

  • This enables expression of mars2 variants to study their functions

  • Fluorescent proteins can be co-expressed to visualize expressing cells

• EnvA-TVA system:

  • EnvA pseudotyped virus specifically infects neurons with promoter-driven expression of TVA

  • This allows for targeting specific cell populations for mars2 studies

  • By driving TVA expression in one hemisphere and injecting EnvA pseudotyped virus into the contralateral hemisphere, neurons with specific projections can be retrogradely labeled

• Manipulation of mars2 activity:

  • Express modified versions of mars2 to study structure-function relationships

  • Co-express activity indicators to monitor effects on mitochondrial function

These tools enable diverse experiments to analyze mars2 expression, function, and role in neural development in Xenopus, making rabies virus a valuable addition to the experimental toolkit .

What insights can be gained from studying mars2 in relation to mitochondrial disease models?

While specific information on mars2 in mitochondrial disease is limited in the search results, we can draw relevant insights from research on related aminoacyl-tRNA synthetases:

• Disease relevance:

  • Dysregulation of mars2 could potentially lead to defects in mitochondrial protein synthesis, affecting production of respiratory chain components

  • This would result in impaired oxidative phosphorylation and energy deficiency, particularly affecting high-energy tissues

• Therapeutic potential:

  • Research on human mitochondrial leucyl tRNA synthetase (LARS2) shows it can suppress defects caused by mutations in non-cognate mt-tRNAs

  • This suggests aminoacyl-tRNA synthetases may have broader functions in supporting mitochondrial translation

  • Similar approaches could be investigated for mars2, exploring whether it can rescue phenotypes associated with mitochondrial tRNA mutations

• C-terminal peptide applications:

  • In human LARS2, a C-terminal peptide alone can enter mitochondria and interact with mt-tRNAs

  • This suggests that small peptides derived from mars2 might similarly be able to interact with and potentially stabilize mutant mt-tRNAs

  • Such peptides could represent novel therapeutic approaches for mitochondrial disorders

• Xenopus as a model system:

  • Xenopus embryos provide an excellent system for studying mitochondrial dysfunction due to their external development

  • Creating Xenopus models with altered mars2 expression could provide insights into disease mechanisms and potential treatments

How does the expression of mars2-like relate to retrotransposon activity during development?

The relationship between mars2-like expression and retrotransposon activity reveals important developmental regulatory mechanisms:

• Coordinated expression patterns:

  • The mars2-like transcript shows an expression profile similar to the retrotransposon λ-olt 2-1

  • Both increase at the blastula stage (stage 9), suggesting common regulatory mechanisms

• Developmental transition:

  • The timing coincides with the maternal-to-zygotic transition, a critical developmental milestone

  • This suggests retrotransposon activity may be specifically regulated during this transition

• Chimeric transcript formation:

  • The mars2-like transcript forms through splicing that connects the LTR of λ-olt 2-1 to mars2-homologous sequences

  • This represents an example of exaptation, where a retrotransposon element has been repurposed

• Transcriptional regulation:

  • Both the retrotransposon λ-olt 2-1 and mars2-like are newly transcribed by RNA polymerase II in embryos, as evidenced by their sensitivity to α-amanitin

  • This indicates active transcriptional control rather than post-transcriptional regulation

• Functional implications:

  • The substitution of conventional mars2 with mars2-like during development suggests selective advantages of this regulatory mechanism

  • This may represent an adaptation for stage-specific control of mitochondrial translation

What experimental approaches can determine if mars2-like produces a functional protein?

Determining whether the mars2-like transcript produces a functional protein requires multiple complementary approaches:

• In vitro translation:

  • Synthesize the mars2-like transcript and test its translation in cell-free systems

  • Compare the products with those of conventional mars2

• Expression constructs:

  • Create expression constructs for both conventional mars2 and mars2-like with epitope tags

  • Express in Xenopus embryos or cell lines and detect protein products by western blotting

• Mass spectrometry:

  • Purify mitochondria from different developmental stages

  • Perform proteomic analysis to identify peptides specific to mars2-like versus conventional mars2

• Functional complementation:

  • Express mars2-like in a system where conventional mars2 has been depleted

  • Test whether mars2-like can rescue the phenotype

• Antibody generation:

  • Generate antibodies specific to the unique N-terminal region of potential mars2-like protein

  • Use these for immunodetection in embryos at different stages

• Ribosome profiling:

  • Perform ribosome profiling on Xenopus embryos at different developmental stages

  • Analyze ribosome occupancy on mars2-like versus conventional mars2 transcripts

• CRISPR-mediated tagging:

  • Use CRISPR-Cas9 to insert a fluorescent tag at the C-terminus of the mars2-like coding sequence

  • Monitor protein expression and localization during development

How might the study of mars2 variants inform our understanding of mitochondrial translation regulation?

The existence of both conventional mars2 and mars2-like transcripts offers unique insights into mitochondrial translation regulation:

• Developmental programming:

  • The switch from conventional mars2 to mars2-like during development suggests stage-specific requirements for mitochondrial translation

  • This may represent a mechanism to adapt protein synthesis to changing energy demands

• Retrotransposon influence:

  • The incorporation of retrotransposon LTR elements into the mars2-like transcript demonstrates how mobile genetic elements can contribute to regulatory diversity

  • This represents a novel mechanism for evolving new gene regulatory patterns

• Maternal-to-zygotic transition:

  • The temporal patterns of expression coincide with the maternal-to-zygotic transition

  • This suggests coordinated regulation between nuclear transcription and mitochondrial translation during this critical developmental window

• Potential protein diversity:

  • If translated, mars2-like would produce a protein with a different N-terminal region

  • This could affect enzyme properties, substrate specificity, or interactions with other cellular components

• Comparative studies:

  • Investigating whether similar mechanisms exist in other species could reveal conserved or divergent strategies for regulating mitochondrial translation

  • This would contribute to our understanding of mitochondrial evolution and adaptation

What therapeutic applications might emerge from research on mars2 and related aminoacyl-tRNA synthetases?

Research on aminoacyl-tRNA synthetases suggests several potential therapeutic applications:

• Suppression of mitochondrial tRNA defects:

  • Research on human mitochondrial leucyl tRNA synthetase (LARS2) demonstrates it can suppress defects caused by mutations in non-cognate mt-tRNAs

  • Similar suppressive effects might be explored for mars2, potentially offering therapeutic approaches for mitochondrial disorders

• Peptide-based therapeutics:

  • The finding that a C-terminal peptide of human LARS2 can enter mitochondria and interact with mt-tRNAs suggests a path toward peptide-based therapies

  • Small peptides derived from mars2 might similarly interact with and stabilize mutant mt-tRNAs

  • Such peptides represent potential therapeutic molecules for pathogenic mt-tRNA mutations

• Gene therapy approaches:

  • Viral vectors expressing mars2 or its functional domains could potentially rescue defects in mitochondrial translation

  • The rabies virus tools demonstrated in Xenopus could potentially be adapted for therapeutic gene delivery

• Cross-species applications:

  • The conservation of aminoacyl-tRNA synthetase functions across species suggests findings in Xenopus mars2 may have relevance to human health

  • Comparative studies could identify conserved functional domains with therapeutic potential

What are the most significant outstanding questions about mars2 in Xenopus?

Despite the insights gained from current research, several important questions about mars2 in Xenopus remain unanswered:

• Protein production:

  • Does the mars2-like transcript produce a functional protein?

  • If so, how does its activity compare to that of conventional mars2?

• Functional significance:

  • What is the biological advantage of having two different mars2 transcripts with distinct temporal expression patterns?

  • Does the switch from conventional mars2 to mars2-like correlate with specific developmental events or metabolic changes?

• Regulatory mechanisms:

  • What factors control the expression of mars2-like and its retrotransposon elements during development?

  • How is the maternal-to-zygotic transition coordinated with changes in mitochondrial translation?

• Species conservation:

  • Is the mars2-like transcript unique to Xenopus, or do similar retrotransposon-derived variants exist in other species?

  • What does this tell us about the evolution of mitochondrial translation systems?

• Disease relevance:

  • Could dysregulation of mars2 variants contribute to developmental disorders or diseases?

  • Might insights from Xenopus mars2 inform our understanding of human mitochondrial diseases?