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Characterization of a ribonuclease III-like protein required for cleavage of the pre-rRNA in the 3'ETS in Arabidopsis.

Comella P, Pontvianne F, Lahmy S, Vignols F, Barbezier N, Debures A, Jobet E, Brugidou E, Echeverria M, Sáez-Vásquez J - Nucleic Acids Res. (2007)

Bottom Line: The modeled 3D structure of the RNaseIII domain of AtRTL2 is similar to the bacterial RNaseIII domain, suggesting a comparable catalytic mechanism.However, unlike bacterial RNaseIII, the AtRTL2 protein forms a highly salt-resistant homodimer that is only disrupted on treatment with DTT.These data indicate that AtRTL2 may use a dimeric mechanism to cleave double-stranded RNA, but unlike bacterial or yeast RNase III proteins, AtRTL2 forms homodimers through formation of disulfide bonds, suggesting that redox conditions may operate to regulate the activity of RNaseIII.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire Génome et Développement des Plantes, UMR 5096 CNRS-UPVD-IRD, Université de Perpignan, 66860 Perpignan cedex, France.

ABSTRACT
Ribonuclease III (RNaseIII) is responsible for processing and maturation of RNA precursors into functional rRNA, mRNA and other small RNA. In contrast to bacterial and yeast cells, higher eukaryotes contain at least three classes of RNaseIII, including class IV or dicer-like proteins. Here, we describe the functional characterization of AtRTL2, an Arabidopsis thaliana RNaseIII-like protein that belongs to a small family of genes distinct from the dicer family. We demonstrate that AtRTL2 is required for 3'external transcribed spacer (ETS) cleavage of the pre-rRNA in vivo. AtRTL2 localizes in the nucleus and cytoplasm, a nuclear export signal (NES) in the N-terminal sequence probably controlling AtRTL2 cellular localization. The modeled 3D structure of the RNaseIII domain of AtRTL2 is similar to the bacterial RNaseIII domain, suggesting a comparable catalytic mechanism. However, unlike bacterial RNaseIII, the AtRTL2 protein forms a highly salt-resistant homodimer that is only disrupted on treatment with DTT. These data indicate that AtRTL2 may use a dimeric mechanism to cleave double-stranded RNA, but unlike bacterial or yeast RNase III proteins, AtRTL2 forms homodimers through formation of disulfide bonds, suggesting that redox conditions may operate to regulate the activity of RNaseIII.

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The A. thaliana genome encodes three RNase III-like proteins that belong to a distinct, non-dicer, gene family. (A) Schematic representation of RNaseIII and RNaseIII-like proteins from A. thaliana (AtRTL1, AtRTL2 and AtRTL3), S. cerevisiae (Rnt1), S. pombe (PacI), E. coli (Ec_RNaseIII) and A. aeolicus (Aa_RNaseIII). Gray boxes correspond to RNaseIII motifs, white boxes to double-strand RNA-binding domain (DS-RBD) and dotted white box in AtRTL1 to less conserved RBD. Black bars correspond to 100 amino acid length. (B) Phylogenetic relation of different RNaseIII and RNase-like proteins. Numbers represent the percentage value of Bootstrap. Accession numbers of sequences used in this analysis: A. thaliana DCL1 (At1g01040), DCL2 (At3g03300), DLC3 (At3g43920), DCL4 (At5g20320), AtRTL1 (this study), AtRTL2 (At3g20420) and AtRTL3 (At5g45150); O. sativa dicer-like proteins (available in the tree), OsRTL1 (Os06g0358800), OsRTL2 (Os05g0271300) and OsRTL3 (Os01g0551100), S. cerevisiae (AAB04172), S. pombe (NP_595292), E. coli (NP_417062), A. aeolicus (NP_213645); C. elegans (NP_501789.1), D. melanogaster (NP_477436.1) and H. sapiens (NP_037367.21).
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Figure 1: The A. thaliana genome encodes three RNase III-like proteins that belong to a distinct, non-dicer, gene family. (A) Schematic representation of RNaseIII and RNaseIII-like proteins from A. thaliana (AtRTL1, AtRTL2 and AtRTL3), S. cerevisiae (Rnt1), S. pombe (PacI), E. coli (Ec_RNaseIII) and A. aeolicus (Aa_RNaseIII). Gray boxes correspond to RNaseIII motifs, white boxes to double-strand RNA-binding domain (DS-RBD) and dotted white box in AtRTL1 to less conserved RBD. Black bars correspond to 100 amino acid length. (B) Phylogenetic relation of different RNaseIII and RNase-like proteins. Numbers represent the percentage value of Bootstrap. Accession numbers of sequences used in this analysis: A. thaliana DCL1 (At1g01040), DCL2 (At3g03300), DLC3 (At3g43920), DCL4 (At5g20320), AtRTL1 (this study), AtRTL2 (At3g20420) and AtRTL3 (At5g45150); O. sativa dicer-like proteins (available in the tree), OsRTL1 (Os06g0358800), OsRTL2 (Os05g0271300) and OsRTL3 (Os01g0551100), S. cerevisiae (AAB04172), S. pombe (NP_595292), E. coli (NP_417062), A. aeolicus (NP_213645); C. elegans (NP_501789.1), D. melanogaster (NP_477436.1) and H. sapiens (NP_037367.21).

Mentions: In order to identify RNaseIII-like proteins in A. thaliana, we performed a Blast search using RNaseIII protein from E. coli and Rnt1 from yeast. Seven genes coding for proteins containing the RNaseIII motif were found in the Arabidopsis genome. Four belong to the DCL family (32,33) and the three others to a new uncharacterized family of RNaseIII genes in Arabidopsis (Figure 1). We initiated the molecular and functional characterization of this new family of genes, namely AtRTL1, AtRTL2 and AtRTL3 (RNAse Three Like1, 2 and 3). The deduced AtRTL1, AtRTL2 and AtRTL3 proteins are 289, 391 and 957 amino acids long with predicted molecular masses of 33, 45 and 108 kDa, respectively. All three sequences contain either one (AtRTL1 and AtRTL2) or two (AtRTL3) RNaseIII domains (Figure 1A). The highly conserved stretch of 9 amino acid residues known as the RNaseIII signature motif (14) is well conserved in AtRTL1 and AtRTL2 sequences but only in the second RNaseIII motif of AtRTL3 (Figure 2B and data not shown). Furthermore, whereas AtRTL2 and AtRTL3 display two and three RNA-binding domains (RBD) respectively, the single putative RBD domain of AtRTL1 seems much less conserved or inexistant (Figure 1A). No other particular protein domains were identified in these three AtRTL sequences. Thus, Arabidopsis RTL proteins do not belong to the Arabidopsis Dicer family which encodes larger proteins (from 1300 to 1900 amino acids long) containing multifunctional domains such as DExHRNA helicase, Piwi/Argonaute/Zwille (PAZ), RNase III and RBD (32,33). Similarly, Arabidopsis RTL protein structure is distinct from human RNaseIII and Drosha proteins that are 1374 and 1327 amino acids long respectively (3,14). Conversely, the Arabidopsis RTL1 and RTL2 proteins show structural similarity to bacterial RNaseIII (15,23), S. cerevisiae Rnt1 (48) and S. pombe Pac1 (17) proteins. However, the N-terminal sequences of RTL1 and RTL2 proteins are longer than bacterial RNaseIII but shorter than Rnt1 and Pac1 yeast RNaseIII-like proteins, which present an additional 100 amino acid sequence in their N-terminal region (Figures 1A and 2B).Figure 1.


Characterization of a ribonuclease III-like protein required for cleavage of the pre-rRNA in the 3'ETS in Arabidopsis.

Comella P, Pontvianne F, Lahmy S, Vignols F, Barbezier N, Debures A, Jobet E, Brugidou E, Echeverria M, Sáez-Vásquez J - Nucleic Acids Res. (2007)

The A. thaliana genome encodes three RNase III-like proteins that belong to a distinct, non-dicer, gene family. (A) Schematic representation of RNaseIII and RNaseIII-like proteins from A. thaliana (AtRTL1, AtRTL2 and AtRTL3), S. cerevisiae (Rnt1), S. pombe (PacI), E. coli (Ec_RNaseIII) and A. aeolicus (Aa_RNaseIII). Gray boxes correspond to RNaseIII motifs, white boxes to double-strand RNA-binding domain (DS-RBD) and dotted white box in AtRTL1 to less conserved RBD. Black bars correspond to 100 amino acid length. (B) Phylogenetic relation of different RNaseIII and RNase-like proteins. Numbers represent the percentage value of Bootstrap. Accession numbers of sequences used in this analysis: A. thaliana DCL1 (At1g01040), DCL2 (At3g03300), DLC3 (At3g43920), DCL4 (At5g20320), AtRTL1 (this study), AtRTL2 (At3g20420) and AtRTL3 (At5g45150); O. sativa dicer-like proteins (available in the tree), OsRTL1 (Os06g0358800), OsRTL2 (Os05g0271300) and OsRTL3 (Os01g0551100), S. cerevisiae (AAB04172), S. pombe (NP_595292), E. coli (NP_417062), A. aeolicus (NP_213645); C. elegans (NP_501789.1), D. melanogaster (NP_477436.1) and H. sapiens (NP_037367.21).
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Figure 1: The A. thaliana genome encodes three RNase III-like proteins that belong to a distinct, non-dicer, gene family. (A) Schematic representation of RNaseIII and RNaseIII-like proteins from A. thaliana (AtRTL1, AtRTL2 and AtRTL3), S. cerevisiae (Rnt1), S. pombe (PacI), E. coli (Ec_RNaseIII) and A. aeolicus (Aa_RNaseIII). Gray boxes correspond to RNaseIII motifs, white boxes to double-strand RNA-binding domain (DS-RBD) and dotted white box in AtRTL1 to less conserved RBD. Black bars correspond to 100 amino acid length. (B) Phylogenetic relation of different RNaseIII and RNase-like proteins. Numbers represent the percentage value of Bootstrap. Accession numbers of sequences used in this analysis: A. thaliana DCL1 (At1g01040), DCL2 (At3g03300), DLC3 (At3g43920), DCL4 (At5g20320), AtRTL1 (this study), AtRTL2 (At3g20420) and AtRTL3 (At5g45150); O. sativa dicer-like proteins (available in the tree), OsRTL1 (Os06g0358800), OsRTL2 (Os05g0271300) and OsRTL3 (Os01g0551100), S. cerevisiae (AAB04172), S. pombe (NP_595292), E. coli (NP_417062), A. aeolicus (NP_213645); C. elegans (NP_501789.1), D. melanogaster (NP_477436.1) and H. sapiens (NP_037367.21).
Mentions: In order to identify RNaseIII-like proteins in A. thaliana, we performed a Blast search using RNaseIII protein from E. coli and Rnt1 from yeast. Seven genes coding for proteins containing the RNaseIII motif were found in the Arabidopsis genome. Four belong to the DCL family (32,33) and the three others to a new uncharacterized family of RNaseIII genes in Arabidopsis (Figure 1). We initiated the molecular and functional characterization of this new family of genes, namely AtRTL1, AtRTL2 and AtRTL3 (RNAse Three Like1, 2 and 3). The deduced AtRTL1, AtRTL2 and AtRTL3 proteins are 289, 391 and 957 amino acids long with predicted molecular masses of 33, 45 and 108 kDa, respectively. All three sequences contain either one (AtRTL1 and AtRTL2) or two (AtRTL3) RNaseIII domains (Figure 1A). The highly conserved stretch of 9 amino acid residues known as the RNaseIII signature motif (14) is well conserved in AtRTL1 and AtRTL2 sequences but only in the second RNaseIII motif of AtRTL3 (Figure 2B and data not shown). Furthermore, whereas AtRTL2 and AtRTL3 display two and three RNA-binding domains (RBD) respectively, the single putative RBD domain of AtRTL1 seems much less conserved or inexistant (Figure 1A). No other particular protein domains were identified in these three AtRTL sequences. Thus, Arabidopsis RTL proteins do not belong to the Arabidopsis Dicer family which encodes larger proteins (from 1300 to 1900 amino acids long) containing multifunctional domains such as DExHRNA helicase, Piwi/Argonaute/Zwille (PAZ), RNase III and RBD (32,33). Similarly, Arabidopsis RTL protein structure is distinct from human RNaseIII and Drosha proteins that are 1374 and 1327 amino acids long respectively (3,14). Conversely, the Arabidopsis RTL1 and RTL2 proteins show structural similarity to bacterial RNaseIII (15,23), S. cerevisiae Rnt1 (48) and S. pombe Pac1 (17) proteins. However, the N-terminal sequences of RTL1 and RTL2 proteins are longer than bacterial RNaseIII but shorter than Rnt1 and Pac1 yeast RNaseIII-like proteins, which present an additional 100 amino acid sequence in their N-terminal region (Figures 1A and 2B).Figure 1.

Bottom Line: The modeled 3D structure of the RNaseIII domain of AtRTL2 is similar to the bacterial RNaseIII domain, suggesting a comparable catalytic mechanism.However, unlike bacterial RNaseIII, the AtRTL2 protein forms a highly salt-resistant homodimer that is only disrupted on treatment with DTT.These data indicate that AtRTL2 may use a dimeric mechanism to cleave double-stranded RNA, but unlike bacterial or yeast RNase III proteins, AtRTL2 forms homodimers through formation of disulfide bonds, suggesting that redox conditions may operate to regulate the activity of RNaseIII.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire Génome et Développement des Plantes, UMR 5096 CNRS-UPVD-IRD, Université de Perpignan, 66860 Perpignan cedex, France.

ABSTRACT
Ribonuclease III (RNaseIII) is responsible for processing and maturation of RNA precursors into functional rRNA, mRNA and other small RNA. In contrast to bacterial and yeast cells, higher eukaryotes contain at least three classes of RNaseIII, including class IV or dicer-like proteins. Here, we describe the functional characterization of AtRTL2, an Arabidopsis thaliana RNaseIII-like protein that belongs to a small family of genes distinct from the dicer family. We demonstrate that AtRTL2 is required for 3'external transcribed spacer (ETS) cleavage of the pre-rRNA in vivo. AtRTL2 localizes in the nucleus and cytoplasm, a nuclear export signal (NES) in the N-terminal sequence probably controlling AtRTL2 cellular localization. The modeled 3D structure of the RNaseIII domain of AtRTL2 is similar to the bacterial RNaseIII domain, suggesting a comparable catalytic mechanism. However, unlike bacterial RNaseIII, the AtRTL2 protein forms a highly salt-resistant homodimer that is only disrupted on treatment with DTT. These data indicate that AtRTL2 may use a dimeric mechanism to cleave double-stranded RNA, but unlike bacterial or yeast RNase III proteins, AtRTL2 forms homodimers through formation of disulfide bonds, suggesting that redox conditions may operate to regulate the activity of RNaseIII.

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