In eukaryotes, seven Sm proteins (SmB, SmD1, 2 and 3, SmE, SmF, and SmG) form a heteroheptameric complex at U-rich Sm binding sites (AU4–6
GR) of various small nuclear RNAs (snRNAs) including the spliceosomal snRNAs U1, U2, U4 and U54,5
. Assembly of Sm proteins in vivo
requires the help of SMN (Survival of Motor Neurons), mutations in which result in spinal muscular atrophy6
. At least two Sm-like complexes have been characterized. The Lsm1-7 complex functions in mRNA degradation7,8
and the Lsm2-8 complex is involved in the maturation of various polymerase III transcripts9–11
and ribosomal RNAs12
. Purified Lsm2-8 binds to the 3′ terminal U-tract on U6, but not to the internal U-rich Sm sites in U1, U2, U4 and U5 snRNAs, illustrating that Sm and Lsm complexes have different sets of target RNAs9
Sm binding sites are also found near the 3′ ends of telomerase RNA subunits from diverse yeasts1,13–16
and are important for RNA processing and/or stability1,2,15
. Actual binding of Sm proteins has been demonstrated for TLC1, the telomerase RNA from S. cerevisiae15
, but the functional consequences of this interaction have remained largely unexplored. The Sm binding site in TLC1 is located several nucleotides upstream of the mature 3′ end13
. In contrast, spliceosomal cleavage of S. pombe
TER1 truncates the putative Sm binding site by one nucleotide2
which may compromise stable association of the Sm ring with mature TER1. We therefore set out to examine which proteins bind to the 3′ end of mature TER1, and to determine the function of the putative Sm site for TER1 biogenesis and stability.
A strategy was developed to examine the 3′ end of TER1 by massively parallel sequencing to obtain a quantitative measure of 3′ end sequence distribution and to identify the most abundant terminal sequences (). This analysis revealed that following spliceosomal cleavage over 60% of TER1 molecules have lost additional nucleotides at the 3′ end and terminate in a stretch of 3 to 6 uridines (). The 3′ end of the majority of TER1 therefore resembles the 3′ end of U6 snRNA, which is bound by the Lsm2-8 complex. To test whether Sm or Lsm proteins associate with TER1, carboxyl-terminal c-Myc epitope tags were inserted at the genomic loci of all Sm and Lsm proteins.
Fig. 1 TER1 RNA associates with Sm and Lsm proteins. (a) Schematic of method used to map the 3′ end distribution of TER1 post spliceosomal cleavage. RNA is depicted in orange, DNA in blue. Abbreviations: PAP, poly(A) polymerase; RT, reverse transcription; (more ...)
Immunoprecipitations were performed with a subset of strains that did not show overt growth defects, expressed tagged proteins and maintained telomeres (Suppl. Fig. 1
). The snRNA U1 control specifically co-precipitated with Sm proteins confirming that the epitope tags did not interfere with immunoprecipitation of RNP complexes (). TER1 co-immunoprecipitated with all four Sm proteins tested (, lanes 2–4 and Suppl. Fig. 2a
), including members of each of the three Sm subcomplexes4
. Strikingly though, several-fold more TER1 was recovered from Lsm IPs resulting in an ~80% depletion of TER1 from the IP supernatant (, lanes 5–7). TER1 precipitated with all subunits of the Lsm2-8 complex ( and Suppl. Fig. 2b
), but not with Lsm1 (, lane 8), the subunit specific to the Lsm1-7 complex.
To determine whether Sm and/or Lsm are associated with active telomerase, direct in vitro
activity assays were performed on immunoprecipitates. Telomerase activity was detected in all samples, but was 20-fold higher in Lsm3 and 4 compared to Smb1 and Sme1 IPs ( and Suppl. Fig. 2c
). In part this can be explained by the lower recovery of telomerase with Sm proteins as judged by quantification of telomerase RNA on northern blots (Suppl. Fig. 2c, d
). However, even after normalization to the amount of TER1 in each IP, Lsm-associated telomerase activity was still 2.8-fold higher than the activity associated with Sm proteins. The simplest explanation for this observation is that a fraction of Sm-associated TER1 is not yet associated with the catalytic subunit of telomerase. Indeed, further experiments confirmed that Sm binding precedes Trt1 binding to TER1.
To gain insights into the functions of Sm and Lsm binding to telomerase we initially focused on the Sm association. For most characterized snRNAs, sequences downstream of the Sm binding site are critical for Sm loading17
. As the mature form of TER1 lacks such sequences, we tested whether the Sm complex was loaded onto the TER1 precursor prior to spliceosomal cleavage. RT-PCR confirmed that the precursor is indeed specifically associated with the Sm complex, but is undetectable in Lsm immunoprecipitates ().
Fig. 2 Sm proteins associate with TER1 precursor and promote spliceosomal cleavage. (a) RNA from α-c-Myc IPs was analysed by RT-PCR using primers in the first and second exon (primers represented by arrows in the schematic below the gel) to amplify the (more ...)
As the spliceosome contains Sm complexes, the TER1-Sm interaction may reflect binding of the spliceosome to the TER1 precursor. To test whether Sm proteins bind TER1 directly, we generated constructs with either a mutant 5′ splice site or a deletion of the intron. Both mutant RNAs co-immunoprecipitated with Smb1 (). In contrast, replacing the Sm binding sequence with a random sequence (ter1-sm6
mutant) reduced Sm association by 22-fold (). Similarly, Lsm association was undetectable for ter1-sm6 (Suppl. Fig. 3a
). We therefore surmised that Sm and Lsm proteins directly bind to the previously identified site in TER1.
We next examined the effect of Sm binding on 3′ end processing by the spliceosome. Loss of Sm binding in the ter1-sm6
mutant resulted in a 7-fold reduction in the processed form (). Furthermore, a series of deletion mutants within the Sm site caused progressive inhibition of TER1 cleavage (Suppl. Fig. 3b
), but not TER1 splicing (Suppl. Fig. 3c
). Finally, introducing an eight-nucleotide spacer between the Sm site and 5′ cleavage site also impaired processing (). In summary, weakening or abolishing Sm association with the TER1 precursor reduces spliceosomal cleavage, indicating that Sm proteins promote 3′ end processing of TER1.
A conserved feature among yeast and mammalian telomerase RNAs is the post-transcriptional hypermethylation of the 5′ cap into a 2,2,7-trimethyl guanosine (TMG) form1,15,18
. Sm proteins were first implicated in promoting cap hypermethylation on U2 snRNA in Xenopus
. It was later shown in vitro
that TMG-capping of human U1 requires the presence of SmB/B′-SmD34,20
. A screen for physical association with Sm proteins led to the identification of Tgs1 in budding yeast21
. To elucidate the roles of Sm and/or Lsm in the hypermethylation of the 5′ cap on TER1, we tested which, if any, of these proteins interact with S. pombe
by two-hybrid. Smd proteins scored positive, with Smd2 displaying the strongest interaction, and the other Sm proteins and all Lsm proteins showing weak or no interaction (Suppl. Fig. 4a
). We next examined whether preventing Sm binding to TER1 affects cap hypermethylation. Whereas wild type TER1 was readily precipitated with a monoclonal antibody against the TMG cap, ter1-sm6 recovery was at least 25-fold reduced ( and Suppl. Fig. 4b
). Only the cleaved form of TER1 was recovered in TMG IPs from wild type cells, suggesting that spliceosomal cleavage precedes hypermethylation (Suppl. Fig. 4c
). TER1 was not TMG-capped in a tgs1Δ
strain confirming that Tgs1 is the enzyme responsible for TER1 cap hypermethylation (Suppl. Fig. 4d
Fig. 3 Tgs1 modifies TER1 and is required for normal telomere maintenance. (a) Loss of Sm site compromises TMG cap formation. RT-PCR amplifying all forms of TER1 and ter1-sm6 from α-TMG IP samples, RT=reverse transcriptase, snRNA U1 served as control. (more ...)
In light of the reported increase in telomerase RNA in tgs1Δ
budding yeast 23
, we were surprised to observe a 5-fold reduction in TER1 RNA in tgs1Δ
compared to wild type in S. pombe
(). In addition, an increase in the precursor indicated a 3′ end processing defect. The viability of tgs1Δ
cells ruled out a major splicing defect, but we consistently noted a small reduction in spliceosomal snRNAs isolated from tgs1Δ
cells ( and data not shown). To differentiate between a processing defect and a direct effect of the TMG cap on TER1 stability, we mutated the spliceosomal cleavage site and inserted a hammerhead ribozyme sequence to generate the mutant ter1-5′ssmut-HH (Suppl. Fig. 4e
). In this construct, processing of TER1 occurs independent of the spliceosome by ribozyme cleavage. When comparing ter1-5′ssmut-HH levels between wild type and tgs1Δ
cells, a 2-fold reduction was observed (). Taken together, these results show that tgs1Δ
affects TER1 processing by the spliceosome as well as TER1 stability. Consistent with the exquisite dosage sensitivity for telomerase RNA in diverse species24,25
, this reduction in TER1 resulted in shorter telomeres (). Neither telomerase activity nor Lsm association was reduced beyond the effects expected from the reduced steady-state level of TER1 (Suppl. Fig. 4f, g
The majority of TER1 post spliceosomal cleavage was bound by Lsm2-8, but a small fraction was associated with Sm proteins (). To investigate whether this was indicative of a switch from Sm to Lsm binding, we examined the distribution of 3′ ends in each IP by massively parallel sequencing. Around 70% of Sm-bound TER1 post-cleavage terminated precisely at the spliceosomal cleavage site ( and Suppl. Fig. 5a
). Enrichment of this form in the Sm-bound fraction is consistent with Sm proteins binding the TER1 precursor and remaining associated with TER1 until after cleavage and cap hypermethylation have occurred. In contrast, Lsm-associated TER1 predominantly terminated in U3–6
indicating that a switch between Sm and Lsm binding occurs after spliceosomal cleavage and is associated with exonucleolytic processing ( and Suppl. Fig. 5b
). Consistent with the majority of telomerase activity being associated with Lsm2-8, the TER1 3′ end distribution from Trt1 immunoprecipitates was indistinguishable from that of Lsm-bound TER1.
Fig. 4 Lsm proteins replace Sm and protect the 3′ end of TER1. (a) 3′ end sequence distribution of TER1 from IP samples. (b) Northern blot analysis from total RNA prepared from wild type, lsm1Δ and lsm3Δ strains, quantified relative (more ...)
The observation that loss of Sm binding coincided with the loss of terminal nucleotides led us to speculate that Lsm2-8 may function in protecting the 3′ end of TER1 against further exonucleolytic degradation. To test this hypothesis we attempted to generate Lsm deletion strains. Whereas most Lsm proteins are essential, lsm1Δ
cells were viable. Consistent with a protective function for Lsm2-8, the levels of TER1 and U6 snRNA were ~5-fold reduced in lsm3Δ
cells (). No such effect was seen when deleting lsm1
, nor was the level of U1 snRNA reduced in lsm3Δ
cells. The 3′ end sequence distribution for TER1 from total RNA of lsm3Δ
cells closely resembled the Sm-bound fraction in wild type, whereas the Lsm-bound fraction was selectively lost in the mutant ( and Suppl. Fig. 5c
). The viability of lsm3Δ
cells further allowed us to confirm that cap hypermethylation is unaffected by the absence of Lsm consistent with Tgs1 acting on TER1 prior to Lsm binding (Suppl. Fig. 5d
To independently verify a role for Lsm proteins in stabilizing TER1 we took advantage of the observation that Lsm binding requires a stretch of consecutive uridines9
. In contrast, Sm binding tolerates other nucleotides in various positions of the binding motif, as exemplified by the Sm binding site in human U1 snRNA (AAUUUGUG). When the TER1 Sm site was mutated to reduce the number of consecutive uridines, the level of mature TER1 was decreased (). We next precipitated Smb1, Lsm4 and Trt1 from wild type and strains containing the ter1-SmU1 mutant. As expected, the mutation had little effect on the binding of Sm proteins (). In fact, when normalized for the lower level of ter1-SmU1 compared to wild type, recovery of ter1-SmU1 with Smb1 was 1.6-fold increased. In contrast, Lsm binding was diminished by more than 20-fold. Most surprisingly, the interaction between the catalytic subunit Trt1 and telomerase RNA was also compromised in the ter1-SmU1 mutant(). The normalized recovery of ter1-SmU1 with Trt1 was 15-fold lower than wild type, indicating that Lsm binding facilitates Trt1 - TER1 association, possibly by inducing a conformational change in the RNA analogous to how binding of the p65 protein facilitates telomerase assembly in Tetrahymena26–28
. Consistent with the poor recovery of ter1-SmU1 in Trt1 immunoprecipitations, in vitro telomerase activity was below the level of detection ().
Fig. 5 Lsm binding to TER1 promotes telomerase assembly and protects TER1 from degradation. (a) Northern blot for TER1. The indicated ratios of mutant to wild type are normalized to the loading control snR101 (LC). (b) Northern blot for TER1 and the ter1-SmU1 (more ...)
Analysis of the 3′ end sequence distribution for ter1-SmU1 from total RNA revealed that most of the mutant RNA ends at the cleavage site(Suppl. Fig. 6
). This form constituted close to 90% of ter1-SmU1 in Smb1 immunoprecipitates. In contrast, Lsm4 and Trt1 IPs predominantly recovered RNA ending in –AUUU and –AUUUG (Suppl. Fig. 6
). These results further support that Trt1 preferentially associates with Lsm-bound telomerase RNA. They also confirm the role of Lsm in protecting the 3′ end of TER1 from further degradation as diminished Lsm binding coincides with an overall reduction in telomerase RNA and a shift towards the form that is bound by Sm.
Taken together our observations demonstrate that distinct populations of TER1 molecules associate with the Sm and Lsm complexes and suggest a sequence of events for TER1 biogenesis (). The polyadenylated TER1 precursor is bound by the Sm complex which promotes spliceosomal cleavage and subsequent 5′ cap hypermethylation by recruiting Tgs1. The Sm ring is then replaced by the Lsm2-8 complex, which protects TER1 from exonucleolytic degradation and promotes binding of the catalytic subunit.
Despite their structural similarity and related binding motifs, Sm and Lsm complexes have different modes of RNA binding and were thought to have distinct and non-overlapping sets of target RNAs. The finding that the TER1 precursor is exclusively associated with the Sm complex, whereas the majority of mature TER1 is bound by Lsm2-8 revealed that biogenesis of telomerase RNA involves both Sm and Lsm complexes. Considering the central roles that Sm and Lsm proteins play in RNA metabolism it will be important to determine whether biogenesis of other non-coding RNAs also involves Sm and Lsm2-8 bound stages. Furthermore, it is interesting to note that several Sm/Lsm proteins have been reported to co-purify with human telomerase (hTR)29,30
raising the possibility that they also function in TMG cap formation and telomerase assembly.