Limits...
A highly conserved protein of unknown function in Sinorhizobium meliloti affects sRNA regulation similar to Hfq.

Pandey SP, Minesinger BK, Kumar J, Walker GC - Nucleic Acids Res. (2011)

Bottom Line: Similar to hfq, smc01113 regulates the accumulation of sRNAs as well as the target mRNAs.Our study provides the first line of evidence for such conceptual parallels.Furthermore, our investigation gives insights into the sRNA-mediated regulation of stress adaptation in S. meliloti.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139-4307, USA.

ABSTRACT
The SMc01113/YbeY protein, belonging to the UPF0054 family, is highly conserved in nearly every bacterium. However, the function of these proteins still remains elusive. Our results show that SMc01113/YbeY proteins share structural similarities with the MID domain of the Argonaute (AGO) proteins, and might similarly bind to a small-RNA (sRNA) seed, making a special interaction with the phosphate on the 5'-side of the seed, suggesting they may form a component of the bacterial sRNA pathway. Indeed, eliminating SMc01113/YbeY expression in Sinorhizobium meliloti produces symbiotic and physiological phenotypes strikingly similar to those of the hfq mutant. Hfq, an RNA chaperone, is central to bacterial sRNA-pathway. We evaluated the expression of 13 target genes in the smc01113 and hfq mutants. Further, we predicted the sRNAs that may potentially target these genes, and evaluated the accumulation of nine sRNAs in WT and smc01113 and hfq mutants. Similar to hfq, smc01113 regulates the accumulation of sRNAs as well as the target mRNAs. AGOs are central components of the eukaryotic sRNA machinery and conceptual parallels between the prokaryotic and eukaryotic sRNA pathways have long been drawn. Our study provides the first line of evidence for such conceptual parallels. Furthermore, our investigation gives insights into the sRNA-mediated regulation of stress adaptation in S. meliloti.

Show MeSH
(A) Directed yeast two hybrid of YbeY. No interaction was detected. (B) Growth of the WT, smc01113 and hfq single, and smc01113 hfq double mutant in LB-MC. Insert shows doubling time of WT and each of the three mutants in LB-MC.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3113577&req=5

Figure 8: (A) Directed yeast two hybrid of YbeY. No interaction was detected. (B) Growth of the WT, smc01113 and hfq single, and smc01113 hfq double mutant in LB-MC. Insert shows doubling time of WT and each of the three mutants in LB-MC.

Mentions: Since our data suggested that the function of SMc01113/YbeY is related to Hfq, we next asked whether SMc01113/YbeY physically interacts with the Hfq protein or with other members of the sRNA machinery and RNA-degradation. The sRNA-machinery and the components of RNA-degradosome are well characterized in E. coli (75). RNase E (rne) has been implicated as an integral component of the sRNA-machinery and degrades targets of sRNAs (10,13,76,77). RNase III (rnc), whose eukaryotic analogs are the DICER/dicer-like (DCL) proteins, participates in sRNA-mediated interaction, along with the RNase E, by performing initial cleavage (78,79). The PIWI domain of the AGO proteins has the RNase H- (rnhB) type of catalytic domain (32). The two other RNases comprising important component of the RNA-degradosome during stress adaptation are RNase R and PNPase; both of these have overlapping functions (80). Informed by the fact that sRNAs can determine which proteins bind to Hfq (81), we asked if we could detect an interaction of YbeY with Hfq and other RNases in the absence of bacterial sRNAs. We therefore conducted a directed yeast two-hybrid screen to determine if YbeY interacts physically with any of these components. However no significant interaction with any of these was observed regardless of whether YbeY was fused with either the GAL4 activation or binding domains (Figure 8). This suggests that either (i) there is no physical interaction between YbeY/SMc01113 with Hfq and other RNases, or (ii) the interaction of YbeY/SMc01113 with these components is highly transient and/or may require additional facilitator proteins. Transient interaction with YbeY, mediated by other proteins would be consistent with the fact that there is a large difference in the number of Hfq protein molecules per cell [approximately 30–60000; (82)] relative to YbeY/SMc01113 (approximately 1000; Walker,G.C., unpublished data) molecules in a cell. The involvement of other co-factors in modulating protein–protein interactions during sRNA regulation is conceivable. For example, it has recently became apparent that the RraA regulatory protein modulates the helicase and RNA-binding activities of the RNA-degradosome (83). Furthermore, the association of the AGO–siRNA complex with AGO/binding proteins e.g. Giw1p for correct localization has been known (84). We also generated a S. meliloti double mutant of hfq and smc01113, which showed highly reduced growth and enhanced doubling time over both the single mutants (Figure 8). As discussed more fully below, these observations are consistent with SMc01113/YbeY and Hfq playing functionally related roles in sRNA regulation but with YbeY playing certain Hfq-independent roles (81) as well.Figure 8.


A highly conserved protein of unknown function in Sinorhizobium meliloti affects sRNA regulation similar to Hfq.

Pandey SP, Minesinger BK, Kumar J, Walker GC - Nucleic Acids Res. (2011)

(A) Directed yeast two hybrid of YbeY. No interaction was detected. (B) Growth of the WT, smc01113 and hfq single, and smc01113 hfq double mutant in LB-MC. Insert shows doubling time of WT and each of the three mutants in LB-MC.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113577&req=5

Figure 8: (A) Directed yeast two hybrid of YbeY. No interaction was detected. (B) Growth of the WT, smc01113 and hfq single, and smc01113 hfq double mutant in LB-MC. Insert shows doubling time of WT and each of the three mutants in LB-MC.
Mentions: Since our data suggested that the function of SMc01113/YbeY is related to Hfq, we next asked whether SMc01113/YbeY physically interacts with the Hfq protein or with other members of the sRNA machinery and RNA-degradation. The sRNA-machinery and the components of RNA-degradosome are well characterized in E. coli (75). RNase E (rne) has been implicated as an integral component of the sRNA-machinery and degrades targets of sRNAs (10,13,76,77). RNase III (rnc), whose eukaryotic analogs are the DICER/dicer-like (DCL) proteins, participates in sRNA-mediated interaction, along with the RNase E, by performing initial cleavage (78,79). The PIWI domain of the AGO proteins has the RNase H- (rnhB) type of catalytic domain (32). The two other RNases comprising important component of the RNA-degradosome during stress adaptation are RNase R and PNPase; both of these have overlapping functions (80). Informed by the fact that sRNAs can determine which proteins bind to Hfq (81), we asked if we could detect an interaction of YbeY with Hfq and other RNases in the absence of bacterial sRNAs. We therefore conducted a directed yeast two-hybrid screen to determine if YbeY interacts physically with any of these components. However no significant interaction with any of these was observed regardless of whether YbeY was fused with either the GAL4 activation or binding domains (Figure 8). This suggests that either (i) there is no physical interaction between YbeY/SMc01113 with Hfq and other RNases, or (ii) the interaction of YbeY/SMc01113 with these components is highly transient and/or may require additional facilitator proteins. Transient interaction with YbeY, mediated by other proteins would be consistent with the fact that there is a large difference in the number of Hfq protein molecules per cell [approximately 30–60000; (82)] relative to YbeY/SMc01113 (approximately 1000; Walker,G.C., unpublished data) molecules in a cell. The involvement of other co-factors in modulating protein–protein interactions during sRNA regulation is conceivable. For example, it has recently became apparent that the RraA regulatory protein modulates the helicase and RNA-binding activities of the RNA-degradosome (83). Furthermore, the association of the AGO–siRNA complex with AGO/binding proteins e.g. Giw1p for correct localization has been known (84). We also generated a S. meliloti double mutant of hfq and smc01113, which showed highly reduced growth and enhanced doubling time over both the single mutants (Figure 8). As discussed more fully below, these observations are consistent with SMc01113/YbeY and Hfq playing functionally related roles in sRNA regulation but with YbeY playing certain Hfq-independent roles (81) as well.Figure 8.

Bottom Line: Similar to hfq, smc01113 regulates the accumulation of sRNAs as well as the target mRNAs.Our study provides the first line of evidence for such conceptual parallels.Furthermore, our investigation gives insights into the sRNA-mediated regulation of stress adaptation in S. meliloti.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139-4307, USA.

ABSTRACT
The SMc01113/YbeY protein, belonging to the UPF0054 family, is highly conserved in nearly every bacterium. However, the function of these proteins still remains elusive. Our results show that SMc01113/YbeY proteins share structural similarities with the MID domain of the Argonaute (AGO) proteins, and might similarly bind to a small-RNA (sRNA) seed, making a special interaction with the phosphate on the 5'-side of the seed, suggesting they may form a component of the bacterial sRNA pathway. Indeed, eliminating SMc01113/YbeY expression in Sinorhizobium meliloti produces symbiotic and physiological phenotypes strikingly similar to those of the hfq mutant. Hfq, an RNA chaperone, is central to bacterial sRNA-pathway. We evaluated the expression of 13 target genes in the smc01113 and hfq mutants. Further, we predicted the sRNAs that may potentially target these genes, and evaluated the accumulation of nine sRNAs in WT and smc01113 and hfq mutants. Similar to hfq, smc01113 regulates the accumulation of sRNAs as well as the target mRNAs. AGOs are central components of the eukaryotic sRNA machinery and conceptual parallels between the prokaryotic and eukaryotic sRNA pathways have long been drawn. Our study provides the first line of evidence for such conceptual parallels. Furthermore, our investigation gives insights into the sRNA-mediated regulation of stress adaptation in S. meliloti.

Show MeSH