Biomedical subjects

Mario Mörl

Publications and source records attributed to Mario Mörl.

8 recordsLinked to original sources

PubMed2006

A splice variant of the human CCA-adding enzyme with modified activity.

The human CCA-adding enzyme (tRNA nucleotidyltransferase) is an essential enzyme that catalyzes the addition of the CCA terminus to the 3' end of tRNA precursors, a reaction which is a fundamental prerequisite for mature tRNAs to become aminoacylated and to participate in protein biosynthesis. To date only one form of this enzyme has been identified in humans. Here, we describe the sequence and activity of a splice variant of the human CCA-adding enzyme identified in public cDNA databases. The in silico analyses performed on this splice variant indicate that there is conservation of the alternative splice donor site among species and indicate that it seems to be used in vivo. Moreover, the recombinantly expressed protein is active in vitro and accepts tRNA transcripts as substrates incorporating the dinucleotide sequence CC to their 3' end, in contrast to the activity of the full length enzyme. These findings strongly suggest that the splice variant of the human CCA-adding enzyme is expressed in the cell although the in vivo function remains unclear.

Adenosine Triphosphate↗

PubMed2005

Is yeast on its way to evolving tRNA editing?

In human mitochondria, genes for tRNA(Tyr) and tRNA(Cys) overlap by a single nucleotide. From polycistronic precursors, a 3'-truncated upstream tRNA(Tyr) is released, missing the overlapping position. A subsequent editing reaction restores this position. Similar mitochondrial tRNA gene overlaps exist in all metazoans, but not in organisms such as yeast or Escherichia coli. Therefore, we asked whether tRNA overlaps are processed in these organisms. Corresponding constructs were introduced and transcripts tested for processing and editing in E. coli and yeast. E. coli produces only one functional tRNA from these precursors, indicating that tRNA overlaps are incompatible with its processing pathway. In contrast, yeast processes overlapping tRNAs similar to human mitochondria, releasing a 3'-truncated upstream tRNA. This tRNA is restored in an editing-like event, although yeast does not carry a corresponding endogenous editing substrate. These findings support the hypothesis of the evolution of editing by recruitment of a pre-existing and promiscuous editing enzyme.

Base Sequence↗

PubMed2004

Mitochondrial tRNA 3' end metabolism and human disease.

Over 150 mutations in the mitochondrial genome have been shown to be associated with human disease. Remarkably, two-thirds of them are found in tRNA genes, which constitute only one-tenth of the mitochondrial genome. A total of 22 tRNAs punctuate the genome and are produced together with 11 mRNAs and 2 rRNAs from long polycistronic primary transcripts with almost no spacers. Pre-tRNAs thus require precise endonucleolytic excision. Furthermore, the CCA triplet which forms the 3' end of all tRNAs is not encoded, but must be synthesized by the CCA-adding enzyme after 3' end cleavage. Amino acid attachment to the CCA of mature tRNA is performed by aminoacyl-tRNA synthetases, which, like the preceding processing enzymes, are nuclear-encoded and imported into mitochondria. Here, we critically review the effectiveness and reliability of evidence obtained from reactions with in vitro transcripts that pathogenesis-associated mutant mitochondrial tRNAs can lead to deficiencies in tRNA 3' end metabolism (3' end cleavage, CCA addition and aminoacylation) toward an understanding of molecular mechanisms underlying human tRNA disorders. These defects probably contribute, individually and cumulatively, to the progression of human mitochondrial diseases.

3' Untranslated Regions↗

PubMed2004

Exchange of regions between bacterial poly(A) polymerase and the CCA-adding enzyme generates altered specificities.

Bacterial poly(A) polymerases (PAP) and tRNA nucleotidyltransferases are highly similar in sequence but display different activities: whereas tRNA nucleotidyltransferase catalyzes the addition of CCA to 3' ends of tRNAs, PAP adds poly(A) tails to a variety of transcripts. Using domain substitution experiments, we show that these enzymes follow a modular concept: exchange of N- and C-terminal regions leads to chimeric enzymes with unexpected activities, indicating that tRNA nucleotidyltransferase carries an "anchor domain" in the C-terminal section that restricts polymerization to three nucleotides. A 27 amino acid region was identified that determines whether poly(A) or CCA is synthesized by the enzyme chimeras. Sequence alignments suggest that the catalytic cores of both enzymes carry identical components involved in nucleotide recognition and incorporation. This seems to be the prerequisite for the observed reprogramming of the catalytic center of PAP to incorporate a sequence of defined length and composition instead of long stretches of A residues.

Amino Acid Motifs↗

PubMed2004

A pathogenesis-associated mutation in human mitochondrial tRNALeu(UUR) leads to reduced 3'-end processing and CCA addition.

Point mutations in mitochondrial tRNAs can cause severe multisystemic disorders such as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) and myoclonus epilepsy with ragged-red fibers (MERRF). Some of these mutations impair one or more steps of tRNA maturation and protein biosynthesis including 5'-end-processing, post-transcriptional base modification, structural stability, aminoacylation, and formation of tRNA-ribosomal complexes. tRNALeu(UUR), an etiologic hot spot for such diseases, harbors 20 of more than 90 disease-associated mutations described to date. Here, the pathogenesis-associated base substitutions A3243G, T3250C, T3271C, A3302G and C3303T within this tRNA were tested for their effects on endonucleolytic 3'-end processing and CCA addition at the tRNA 3'-terminus. Whereas mutations A3243G, A3302G and C3303T reduced the efficiency of 3'-end cleavage, only the C3303T substitution was a less efficient substrate for CCA addition. These results support the view that pathogenesis may be elicited through cumulative effects of tRNA mutations: a mutation can impede several pre-tRNA processing steps, with each such reduction contributing to the overall impairment of tRNA function.

Humans↗

PubMed2004

The tRNA world.

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Animals↗

PubMed2003

Crystal structure of the human CCA-adding enzyme: insights into template-independent polymerization.

All tRNA molecules carry the invariant sequence CCA at their 3'-terminus for amino acid attachment. The post-transcriptional addition of CCA is carried out by ATP(CTP):tRNA nucleotidyltransferase, also called CCase. This enzyme catalyses a unique template-independent but sequence-specific nucleotide polymerization reaction. In order to reveal the molecular mechanism of this activity, we solved the crystal structure of human CCase by single isomorphous replacement. The structure reveals a four domain architecture with a cluster of conserved residues forming a positively charged cleft between the first two domains. Structural homology of the N-terminal CCase domain to other nucleotidyltransferases could be exploited for modeling a tRNA-substrate complex. The model places the tRNA 3'-end into the N-terminal nucleotidyltransferase site, close to a patch of conserved residues that provide the binding sites for CTP and ATP. Based on our results, we introduce a corkscrew model for CCA addition that includes a fixed active site and a traveling tRNA-binding region formed by flexible parts of the protein.

Amino Acid Sequence↗

PubMed2002

A universal method to produce in vitro transcripts with homogeneous 3' ends.

A method is described that allows a general drawback of in vitro transcription assays to be overcome: RNA polymerases tend to add extra nucleotides to the RNA 3' end that are not encoded in the linearized DNA template. Furthermore, these polymerases show a considerable rate of premature termination close to the RNA's 3' end. These features lead to a decreased yield of full-length transcripts and often make it difficult to determine and isolate the correctly transcribed full-length RNA. The hammerhead ribozyme is frequently used in cis to cleave off these extra nucleotides. However, the upstream sequence requirements of this ribozyme restrict its general usability. In contrast, the hepatitis delta virus ribozyme has no such requirements and can therefore be applied to any RNA sequence in cis. Due to the catalytic activity of the ribozyme, the desired transcript is released as an RNA molecule with a homogeneous 3' end. The resulting 2',3'-cyclo-phosphate group of the released RNA can be easily and efficiently removed by T4 polynucleotide kinase treatment. The presented method can be applied for virtually any sequence to be transcribed and is therefore superior to other ribozyme strategies, suggesting possible applications in every field where transcripts with homogeneous 3' ends are required.

Animals↗