Two complexes of Lsm proteins, Lsm1–Lsm7 and Lsm2–Lsm8, have been described in yeast cells. The Lsm1–Lsm7 complex functions in mRNA decay, whereas the Lsm2–Lsm8 complex is important for U6 snRNA stability and function. We found that a third Lsm complex, composed of Lsm2–Lsm7, associates with about half of the box H/ACA snoRNA snR5 in Saccharomyces cerevisiae. Using live cell imaging, we determined that Lsm proteins are present in nucleoli. Together with previous data that Lsm2–Lsm7 proteins bind pre-RNase P RNA, our data suggest that an Lsm2–Lsm7 complex plays a role in the biogenesis or function of a subset of nucleolus-associated small RNAs.
Although all well-characterized eukaryotic Sm and Lsm protein complexes contain seven subunits, only six Lsm proteins associate with snR5 and pre-RNase P RNA. One possibility is that an unidentified yeast protein, whose homology to Lsm proteins has escaped detection, forms the seventh member of the complex. Alternatively, one Lsm protein could be present in two copies, resulting in a seven-membered ring. Finally, as described for Hfq (Moller et al., 2002
; Zhang et al., 2002
), the snR5-bound Lsm complex may be a hexamer. Consistent with a hexamer, only six proteins were identified in the Xenopus
Lsm complex that binds U8 snoRNA (Tomasevic and Peculis, 2002
The finding that both RNAs that bind Lsm2–Lsm7, snR5, and pre-RNase P RNA, are nucleolar (Srisawat et al., 2002
; see also ) suggests that the Lsm2–Lsm7 complex functions in nucleoli. For the Lsm1–Lsm7 and Lsm2–Lsm8 complexes, Lsm1p and Lsm8p are likely responsible for the different subcellular locations of their respective complexes. Similarly, Lsm10, a member of the Sm complex that binds U7 snRNA, may govern localization of U7 to coiled bodies (Pillai et al., 2001
). Thus, an unidentified seventh Lsm protein could confer nucleolar localization of the Lsm2–Lsm7 complex. Alternatively, the default subcellular location for Lsm proteins may be nucleoli, with Lsm1p or Lsm8p required to localize the complex elsewhere in cells. As a different set of six Lsm proteins (Lsm2–Lsm4 and Lsm6–Lsm8) were identified in the complex that binds Xenopus
U8 RNA (Tomasevic and Peculis, 2002
), another possibility is that any complex containing only six Lsm proteins will localize to nucleoli.
Our finding that binding of Lsm proteins to snR5 requires the RNA 3′ end is consistent with the fact that Lsm2–Lsm8 and Lsm1–Lsm7 likely associate with the 3′ ends of their target RNAs. The 3′ end of U6 snRNA is protected by Lsm2–Lsm8 proteins from nucleases (Achsel et al., 1999
) and is required for Lsm protein binding in vitro (Vidal et al., 1999
). Similarly, the presence of 3′ trimmed mRNAs in lsm
mutant strains suggests that Lsm1–Lsm7 stabilizes the 3′ ends of deadenylated mRNAs from exonucleases (He and Parker, 2001
). To date, the sole exception is the Lsm complex that associates with U8 snoRNA, which requires an internal sequence for efficient binding in vitro (Tomasevic and Peculis, 2002
). One possibility is that Lsm complexes play a general role in stabilizing associated RNAs from nucleases. Our finding that snR5 is stable during Lsm depletion does not negate this possibility, because other proteins may redundantly stabilize this RNA. A role in stabilizing 3′ ends would be reminiscent of the La protein, which stabilizes the 3′ ends of nascent RNAs ending in UUUOH
(Wolin and Cedervall, 2002
). However, our finding that additional RNAs do not become bound by Lsm proteins in strains lacking Lhp1p () indicates that binding of these proteins to RNA 3′ ends is not interchangeable.
How many roles do Lsm complexes play in eukaryotic cells? In bacteria, the Sm-like protein Hfq binds a myriad of small RNAs, stabilizing them from degradation, and facilitating basepairing between many of these RNAs and their targets. Hfq is also implicated in the translation and degradation of several mRNAs (reviewed by Masse et al., 2003
). In yeast, Lsm2–Lsm8 and Lsm1–Lsm7 function in U6 metabolism and mRNA decay, respectively. Because abnormalities in pre-tRNA processing, rRNA processing and U3 snoRNA maturation occur on depletion of essential Lsm proteins, Lsm proteins have also been proposed to function in these processes (Kufel et al., 2002
). However, as overexpression of U6 snRNA suppresses many of the detected changes in processing (), these defects may be secondary to the decline in U6 snRNA levels. Nonetheless, our finding that Lsm2–Lsm7 associate with about half the snR5 in yeast, coupled with the finding that Xenopus
Lsm proteins bind U8 RNA (Tomasevic and Peculis, 2002
), implies an additional function for these proteins in the biogenesis or function of at least a subset of nucleolar RNAs.
What role might Lsm proteins play in snoRNA metabolism? One possibility is that Lsm proteins assist specific snoRNAs in basepairing with their rRNA targets. This role would be consistent with the finding that Hfq assists RNA-RNA pairing (Moller et al., 2002
; Zhang et al., 2002
) and the fact that most small RNAs bound by Sm family members function by basepairing with other RNAs. In this scenario, the fact that rRNA pseudouridylation is unaffected by Lsm protein depletion may reflect redundancy between box H/ACA-specific proteins and Lsm proteins in assisting basepairing. However, although snR5 and U8 snoRNAs base pair with rRNA (Ganot et al., 1997
; Tomasevic and Peculis, 2002
), basepairing has not been described between RNase P and pre-tRNAs. It is also unclear why snR5 and U8, and not the many other snoRNAs that base pair with rRNA, would require Lsm-assisted basepairing. One possibility is that the Lsm2–Lsm7 complex interacts transiently with many snoRNAs to facilitate basepairing, but associates stably with snR5, perhaps because the 3′ end of snR5 contains a high-affinity site for Lsm protein binding.
A second possibility is that the Lsm2–Lsm7 complex assists in the biogenesis of snR5, RNase P, and perhaps additional snoRNAs. A role in snoRNA biogenesis is suggested by the finding that LSM5
has genetic interactions with SRP40
, a gene implicated in both box H/ACA and box C/D snoRNA biogenesis (Yang and Meier, 2003
). Possible roles include localization or retention of these snoRNAs in nucleoli and facilitation of RNA maturation or RNP assembly.
Finally, the Lsm2–Lsm7 complex may assist snR5, and perhaps pre-RNase P, in functions that are distinct from their roles in rRNA pseudouridylation and pre-tRNA maturation. Consistent with this possibility, our experiments suggest that the Lsm-associated snR5 is at least partly nonoverlapping with the complex formed by snR5 RNA with the box H/ACA-specific proteins Gar1 and Nhp2. Moreover, the fact that Lsm proteins require the last 9 nt of snR5, which contain the conserved ACA box, hints that the binding of box H/ACA snoRNP proteins and Lsm proteins may be mutually exclusive. Interestingly, our gradient fractionations reveal that the Lsm-associated snR5 RNA sediments in complexes ranging from ~18S to greater than 25S, suggesting that additional proteins or RNAs are present. Identification of these other components may give insights into the role of the Lsm2–Lsm7/snR5 RNA complex in cell function.