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Category: Microbial Genetics and Molecular Biology
Mitochondrial mRNA Editing in Kinetoplastid Protozoa, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555818296/9781555811334_Chap21-1.gif /docserver/preview/fulltext/10.1128/9781555818296/9781555811334_Chap21-2.gifAbstract:
The mRNAs in the mitochondrion of kinetoplastid protozoa are often modified by the precise addition or deletion of uridylates (U's) by a process termed RNA editing. This chapter focuses on the molecular basis of RNA editing, and the integration of biochemical mechanism with the structure of the editing machinery. The mitochondrial mRNAs are translated on mitochondrial ribosomes and produce functional proteins. It is clear that while minicircles do not encode proteins directly, they play an important role in RNA maturation by encoding most of the guide RNAs (gRNAs) necessary for the editing of mitochondrial mRNAs. The secondary structures determined for the four gRNAs, while similar, have Gibbs free energies well below those predicted for the most stable structures for these RNAs. In a series of UV cross-linking studies, gRNAs binding proteins have also been identified from Trypanosoma brucei mitochondrial extracts. When different gRNAs specific to different mRNA editing sites are incubated with mitochondrial extracts, the same eight proteins are able to bind. Based entirely on analysis of genomic and cDNA sequences, we can predict with some certainty that RNA editing is obligatory for the formation of mitochondrial mRNAs with correct initiation and termination codons and continuous open reading frames to encode these mitochondrial proteins.
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Transcription maps of the maxicircle and minicircle of T. brucei. The upper part depicts the linear map of the 23,019 bp maxicircle ( Sloof et al., 1992 ) of T. brucei. The genes above the line are transcribed from left to right, while the genes beneath the line are transcribed from right to left. The ribosomal RNAs (12S and 9S) have added U's at their 3′ ends ( Adler et al., 1991 ). Transcripts from cytochrome b (CYb), cytochrome oxidase II (COII) and maxicircle unidentified reading frame (MURF) 2 have limited amounts of internal editing (black diamonds). The transcripts from the genes encoding NADH dehydrogenase 3, 7, 8, and 9 (ND3, ND7, ND8, and ND9), cytochrome oxidase III (COIII), ATPase subunit 6 (A6), small subunit ribosomal protein S12, and GC-rich regions 3 and 4 (CR3 and CR4) are all extensively edited (shaded boxes). The variable region of the maxicircle is indicated (VR). In the lower part, a linear map of a 1 kb minicircle of T. brucei is given. The bent helical region of this minicircle (open box) and the origin of replication (ori) are within the conserved region of the minicircle. The gRNA genes (arrows) are flanked by 18 bp imperfect inverted repeat sequences (dark boxes). (Adapted from Hajduk and Sabatini, 1996. )
Transcription maps of the maxicircle and minicircle of T. brucei. The upper part depicts the linear map of the 23,019 bp maxicircle ( Sloof et al., 1992 ) of T. brucei. The genes above the line are transcribed from left to right, while the genes beneath the line are transcribed from right to left. The ribosomal RNAs (12S and 9S) have added U's at their 3′ ends ( Adler et al., 1991 ). Transcripts from cytochrome b (CYb), cytochrome oxidase II (COII) and maxicircle unidentified reading frame (MURF) 2 have limited amounts of internal editing (black diamonds). The transcripts from the genes encoding NADH dehydrogenase 3, 7, 8, and 9 (ND3, ND7, ND8, and ND9), cytochrome oxidase III (COIII), ATPase subunit 6 (A6), small subunit ribosomal protein S12, and GC-rich regions 3 and 4 (CR3 and CR4) are all extensively edited (shaded boxes). The variable region of the maxicircle is indicated (VR). In the lower part, a linear map of a 1 kb minicircle of T. brucei is given. The bent helical region of this minicircle (open box) and the origin of replication (ori) are within the conserved region of the minicircle. The gRNA genes (arrows) are flanked by 18 bp imperfect inverted repeat sequences (dark boxes). (Adapted from Hajduk and Sabatini, 1996. )
Genomic organization of minicircle gRNA genes. (A) Linear map of the minicircle of T. brucei (see Fig. 1 ). The transcript Tl is probably a primer for DNA replication. (B) A single gRNA transcription unit, showing the 18 bp repeats, start site for transcription and spacing between upstream repeat and transcription start site. (C) General features of gRNAs. 5ppp indicates that gRNAs are primary transcripts, the anchor base pairs with the preedited mRNA, the guide sequence directs U addition or deletion, and the 3′ oligo(U) tail is added posttranscriptionally. (Adapted from Hajduk and Sabatini, 1996. )
Genomic organization of minicircle gRNA genes. (A) Linear map of the minicircle of T. brucei (see Fig. 1 ). The transcript Tl is probably a primer for DNA replication. (B) A single gRNA transcription unit, showing the 18 bp repeats, start site for transcription and spacing between upstream repeat and transcription start site. (C) General features of gRNAs. 5ppp indicates that gRNAs are primary transcripts, the anchor base pairs with the preedited mRNA, the guide sequence directs U addition or deletion, and the 3′ oligo(U) tail is added posttranscriptionally. (Adapted from Hajduk and Sabatini, 1996. )
Proposed structure of a gRNA hybridized to preedited and edited mRNA. Two distinct duplexes are formed between preedited mRNA and its cognate gRNA. An “anchor” duplex exists where the 5′ end of the gRNA hybridizes to the preedited RNA immediately 3′ to the first editing site. The first base pair mismatch of this duplex may direct the editing site-specific endonuclease reaction. A second duplex exists between the oligo(U) tail of the gRNA and the purine rich region 5′ to the editing site. This duplex may serve to hold the 5′ cleavage fragment after endonuclease cleavage occurs. The sequence of the gRNA between these two duplexes directs the editing at sites 1 through 4. Editing is complete when the gRNA is able to hybridize throughout its entire region with the mRNA.
Proposed structure of a gRNA hybridized to preedited and edited mRNA. Two distinct duplexes are formed between preedited mRNA and its cognate gRNA. An “anchor” duplex exists where the 5′ end of the gRNA hybridizes to the preedited RNA immediately 3′ to the first editing site. The first base pair mismatch of this duplex may direct the editing site-specific endonuclease reaction. A second duplex exists between the oligo(U) tail of the gRNA and the purine rich region 5′ to the editing site. This duplex may serve to hold the 5′ cleavage fragment after endonuclease cleavage occurs. The sequence of the gRNA between these two duplexes directs the editing at sites 1 through 4. Editing is complete when the gRNA is able to hybridize throughout its entire region with the mRNA.
Schematic drawing of the cleavage-ligation mechanism for insertional RNA editing. An edited cytochrome b mRNA is formed by the sequential formation of the binary gRNA-mRNA structure (step 1). This is followed by endonuclease cleavage of the pre-mRNA at the editing site immediately adjacent to the duplex gRNA-mRNA anchor (step 2). The 5′ fragment of the pre-mRNA contains a 3′ terminal hydroxy 1 which is the substrate for the addition of U's by the mitochondrial TUTase (step 3). Following U addition the cleavage fragments are joined by the mitochondrial RNA ligase to form the edited mRNA product (step 4).
Schematic drawing of the cleavage-ligation mechanism for insertional RNA editing. An edited cytochrome b mRNA is formed by the sequential formation of the binary gRNA-mRNA structure (step 1). This is followed by endonuclease cleavage of the pre-mRNA at the editing site immediately adjacent to the duplex gRNA-mRNA anchor (step 2). The 5′ fragment of the pre-mRNA contains a 3′ terminal hydroxy 1 which is the substrate for the addition of U's by the mitochondrial TUTase (step 3). Following U addition the cleavage fragments are joined by the mitochondrial RNA ligase to form the edited mRNA product (step 4).
Mitochondrial editing complex-associated protein 1 (REAP-1). Schematic drawing showing the general features of REAP-1. The sequence and relative position of the positively charged 21 amino acid repeats (positively charged residues are underlined) and a putative mitochondrial targeting sequence are shown. The role of the targeting sequence in mitochondrial localization has not been established.
Mitochondrial editing complex-associated protein 1 (REAP-1). Schematic drawing showing the general features of REAP-1. The sequence and relative position of the positively charged 21 amino acid repeats (positively charged residues are underlined) and a putative mitochondrial targeting sequence are shown. The role of the targeting sequence in mitochondrial localization has not been established.
Proposed model for assembly and maturation of editing complexes. gRNAs are associated with specific proteins in a 19S RNP which is the initial site of posttranscriptional U addition to the gRNAs. The 19S RNP can bind pre-mRNAs and associated proteins to form a 35–40S RNP. The assembly of 35-WS RNP probably requires base pairing of the anchor region of the gRNA and mRNA immediately 3′ to the preedited region (PER) of the mRNA. The complex may function to hold the 5′ mRNA fragment in position for the second step in editing. Specific proteins of 9, 21, and 90 kDa have been identified by UV cross-linking ( Goringer et al., 1994 ), while RNA ligase, TUTase, and endonuclease activities have been assayed directly ( Pollard et al., 1992 ; Sabatini and Hajduk, 1995 ).
Proposed model for assembly and maturation of editing complexes. gRNAs are associated with specific proteins in a 19S RNP which is the initial site of posttranscriptional U addition to the gRNAs. The 19S RNP can bind pre-mRNAs and associated proteins to form a 35–40S RNP. The assembly of 35-WS RNP probably requires base pairing of the anchor region of the gRNA and mRNA immediately 3′ to the preedited region (PER) of the mRNA. The complex may function to hold the 5′ mRNA fragment in position for the second step in editing. Specific proteins of 9, 21, and 90 kDa have been identified by UV cross-linking ( Goringer et al., 1994 ), while RNA ligase, TUTase, and endonuclease activities have been assayed directly ( Pollard et al., 1992 ; Sabatini and Hajduk, 1995 ).
mRNAs in T. brucei
mRNAs in T. brucei