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The splicing of precursor tRNAs (pre-tRNAs) is an essential step in tRNA maturation, ensuring their functionality in translation. Eukaryotic tRNA splicing endonucleases catalyze this process, cleaving intron-containing pre-tRNAs to generate mature tRNAs. Despite its fundamental importance, the molecular mechanisms underlying tRNA splicing endonucleases have remained enigmatic until recent years. This review aims to elucidate the latest breakthroughs in deciphering the eukaryotic tRNA splicing endonuclease machinery [1].

Structural insights

Recent structural studies have provided unprecedented insights into the architecture of eukaryotic tRNA splicing endonucleases. High-resolution cryo-electron microscopy (cryo-EM) and X-ray crystallography have revealed the intricate arrangements of protein subunits within the endonuclease complex [2]. These structural elucidations shed light on the catalytic mechanism and substrate recognition of tRNA splicing endonucleases, unraveling the molecular basis of their specificity and efficiency.

Functional characterization

Functional characterization studies have elucidated the biochemical properties and regulatory mechanisms governing eukaryotic tRNA splicing endonucleases [3]. Through biochemical assays and mutagenesis studies, researchers have delineated the roles of individual protein subunits and cofactors in endonuclease activity and substrate recognition. Moreover, kinetic analyses have provided insights into the kinetics of tRNA splicing, unveiling the intricacies of the catalytic mechanism and its modulation under different cellular conditions.

Evolutionary perspectives

Comparative genomics and phylogenetic analyses have offered intriguing insights into the evolutionary history of tRNA splicing endonucleases across diverse taxa. By examining the conservation patterns of endonuclease subunits and their associated domains, researchers have traced the evolutionary trajectories of these essential enzymes. Furthermore, evolutionary studies have uncovered the origins of tRNA splicing endonucleases and their co-evolutionary relationships with tRNA genes, shedding light on the evolutionary forces driving their diversification and adaptation [4].

Architecture of the human TSEN complex

The architecture of the human tRNA splicing endonuclease (TSEN) complex is a marvel of molecular machinery intricately designed to execute the essential task of cleaving intron-containing precursor tRNAs (pre-tRNAs) during tRNA maturation. Comprising four subunits—TSEN2, TSEN15, TSEN34, and TSEN54—the human TSEN complex forms a stable heterotetrameric assembly critical for its endonuclei.

Structure of intron-containing tRNAs

Intron-containing tRNAs exhibit a unique structural arrangement characterized by the presence of one or more intervening sequences, known as introns, within their primary sequence. These introns are non-coding regions that must be removed through splicing to yield mature, functional tRNAs. Here are some key points regarding the structure of intron-containing tRNAs:

Primary structure: The primary structure of intron-containing tRNAs consists of conserved elements essential for their function in protein synthesis. These elements include the acceptor stem, D stem-loop, anticodon loop, and TψC stem-loop. Additionally, the intron interrupts the sequence between the anticodon loop and the TψC stem-loop.

Secondary structure: Despite the presence of introns, intron-containing tRNAs fold into the characteristic cloverleaf secondary structure typical of all tRNAs. This structure comprises four arms: the acceptor stem, D stem-loop, anticodon loop, and TψC stem-loop [5]. The intron forms an additional loop between the anticodon loop and the TψC stem-loop, creating a distinctive bulge in the cloverleaf structure.

Tertiary structure: The tertiary structure of intron-containing tRNAs involves intricate interactions between different regions of the molecule, including base pairing and stacking interactions. The intron forms specific tertiary contacts with surrounding regions of the tRNA molecule, influencing its overall folding and stability.

Splicing signals: Intron-containing tRNAs contain specific sequence motifs known as splice sites that are recognized by the splicing machinery during the removal of introns. These signals typically consist of conserved sequences located at the boundaries between exons and introns, facilitating precise cleavage and ligation reactions during splicing.

Modifications: Intron-containing tRNAs undergo post-transcriptional modifications, similar to their intron-less counterparts, to ensure proper function in translation. These modifications often occur at specific nucleotide positions within the tRNA molecule and may be influenced by the presence of introns.

Functional implications: The presence of introns in tRNAs introduces additional complexity into the process of tRNA maturation. Splicing of introns is essential for generating mature, functional tRNAs capable of participating in protein synthesis. Any disruptions or errors in the splicing process can lead to defects in translation and potentially impact cellular function.

Therapeutic implications

The elucidation of the tRNA splicing endonuclease machinery has profound implications for therapeutic interventions targeting RNA processing-related disorders. Dysregulation of tRNA splicing has been implicated in various human diseases, including neurological disorders and cancer. Therefore, understanding the molecular mechanisms underlying tRNA splicing endonucleases holds promise for the development of novel therapeutic strategies, such as small molecule inhibitors and gene editing technologies, to rectify aberrant RNA processing and restore cellular homeostasis [6-9].

Conclusion

In conclusion, recent advancements in understanding the eukaryotic tRNA splicing endonuclease machinery have unveiled the intricate molecular mechanisms governing tRNA splicing. Structural, functional, and evolutionary insights have provided a comprehensive understanding of these essential enzymes, paving the way for future research endeavors and therapeutic interventions targeting RNA processing-related disorders. Further exploration of the tRNA splicing endonuclease machinery promises to unravel additional layers of complexity in RNA processing and gene expression regulation.

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