Pof8 is a La-related protein and a constitutive component of telomerase in fission yeast
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Pof8 is a La-related protein and a constitutive component of telomerase in fission yeast
Diego J. P?ez-Moscoso 0
Lili Pan 0
Rutendo F. Sigauke 0
Morgan R. Schroeder 0
Wen Tang 0 4
Peter Baumann 0 1 2 3
0 Stowers Institute for Medical Research , Kansas City, MO 64110 , USA
1 Howard Hughes Medical Institute , Kansas City, MO 64110 , USA
2 Department of Molecular and Integrative Physiology, University of Kansas Medical Center , Kansas City, MO 66160 , USA
3 Institute of Developmental Biology and Neurobiology, Johannes Gutenberg University , 55099 Mainz , Germany
4 Present address: RNA Therapeutics Institute, University of Massachusetts Medical , USA
Telomerase reverse transcriptase (TERT) and the non-coding telomerase RNA subunit (TR) constitute the core of telomerase. Here we now report that the putative F-box protein Pof8 is also a constitutive component of active telomerase in fission yeast. Pof8 functions in a hierarchical assembly pathway by promoting the binding of the Lsm2-8 complex to telomerase RNA, which in turn promotes binding of the catalytic subunit. Loss of Pof8 reduces TER1 stability, causes a severe assembly defect, and results in critically short telomeres. Structure profile searches identified similarities between Pof8 and telomerase subunits from ciliated protozoa, making Pof8 next to TERT the most widely conserved telomerase subunits identified to date.
I composed of short tandem repeat sequences maintained by the
n most eukaryotes, the DNA component of telomeres is
reverse transcriptase telomerase. Telomerase is a
ribonucleoprotein (RNP) complex in which the RNA subunit (TR, TER1 in
Schizosaccharomyces pombe) functions as a scaffold for the
assembly of telomerase reverse transcriptase (TERT, Trt1 in S.
pombe) and other protein subunits1. The RNA subunit also
contains the template region for telomere repeat synthesis. In all
species examined, telomerase RNA subunits are transcribed as
precursors that then undergo a series of processing events to
produce the mature form that is assembled into the active
telomerase complex. In S. pombe, the mature form of TER1 is ~1213
nucleotides in length2,3 and ends just upstream of a 5? splice site4.
The precursor is about 200 nucleotides longer, containing an
intron and second exon. Interestingly, TER1 maturation involves
only the first step of a splicing reaction. After spliceosomal
cleavage at the 5? splice site, the first exon is released to become
the mature form of telomerase. This reaction is favored by RNA
elements within TER1 that promote a slow transition between the
two steps of splicing resulting in the ?discard? of splicing
intermediates5. A role for spliceosomal cleavage in 3? end processing of
telomerase is conserved among many fungi, but the underlying
mechanisms are surprisingly diverse6,7.
Fungal telomerase RNAs contain an Sm-binding site near the
mature 3? end, and require binding of the hetero-heptameric Sm
complex for processing and stability4,8?10. The Sm complex is a
member of the Hfq-family of RNA-binding proteins that is
conserved in all domains of life11. Sm proteins also assemble on
spliceosomal small nuclear RNAs (snRNAs), where they are
critical for 5? cap hypermethylation, reimport of the RNPs into the
nucleus and spliceosome function12. During S. pombe TER1
maturation, Sm proteins promote spliceosomal cleavage and
recruit the methyl transferase Tgs1 that generates the
2,2,7-trimethylguanosine (TMG) cap8. The Sm complex then dissociates
from TER1 and is replaced by the Sm-like complex Lsm2-8,
which protects the 3? end of TER1 from degradation and
promotes the association of TER1 with Trt1 to generate the
Biochemical and structural studies of telomerases from
ciliated protozoa has provided fundamental insights into
telomerase biogenesis and function. However, the extent to which
these findings can inform studies in other organisms has
remained less clear due to fundamental differences in enzyme
composition and biogenesis pathways. For example, the
telomerase RNA subunit is transcribed by RNA polymerase III in
ciliates, but by RNA pol II in yeasts and metazoans. It has
thus been thought that proteins involved in the processing
and stabilization of pol III transcripts may only function in
telomerase RNA biogenesis in ciliates. This includes members of
the Lupus La antigen-related protein (LARP) family13, which are
components of the telomerase holoenzyme in ciliates and are
critical for the assembly, nuclear retention, and activity of
We now demonstrate that a critical role for La family members
in telomerase biogenesis and function is conserved in
fission yeast, where telomerase RNA is a pol II transcript.
Our results reveal that the Pof8 protein binds to telomerase
RNA, functions in hierarchical assembly by promoting Lsm2-8
binding, and forms a constitutive component of the active
enzyme. Profile database searches identify Pof8 as a previously
unrecognized member of the LARP family displaying striking
structural similarities with human and ciliate proteins.
Our findings reveal an ancient role for La-related proteins
(LARP) in telomerase biogenesis and indicate that evolutionary
conservation in holoenzyme composition extends much further
than previously thought.
Pof8 is a La-related protein family member. To gain a better
understanding of the transition from Sm protein-bound TER1
precursor to the Lsm-bound mature form, we performed
immunoprecipitations for each of the Sm and Lsm proteins from
strains with myc epitope tags on individual subunits8. Precipitates
were used to identify associated proteins and RNAs by mass
spectrometry and Illumina sequencing, respectively. Our
attention focused on the Pof8 protein as it was reliably precipitated by
Lsm2 and Lsm8 and not found in control IPs. Originally reported
as a putative F-box protein19, Pof8 had previously been
implicated in telomere maintenance by screening the S. pombe gene
deletion collection for strains with abnormal telomere length20.
Using sequence- and profile-based searches, we were unable to
independently confirm the F-box domain previously described21.
However, a homology search readily identified an RNA
recognition motif (RRM) near the C-terminus of Pof8 (Fig. 1a, b). This
RRM most closely resembled RRMs in the human LARP family.
A subsequent profile sequence search of the full-length
Pof8 sequence using HHpred22 revealed a La motif and an
additional RRM (Fig. 1a, c). Both domains independently
identified Pof8 as a LARP, the same family that includes the
telomerase subunits p65 from Tetrahymena thermophila15 and p43
from Euplotes aediculatus14.
Reduced TER1 level and short telomeres in pof8? cells. The
sequence similarity with bona fide telomerase components in
ciliates, combined with the interaction of Pof8 with Lsm proteins,
lead us to hypothesize that Pof8 may be directly involved in
telomerase biogenesis. Examination of TER1 by northern blotting
revealed a four to five-fold reduction in RNA levels in pof8? cells
(Fig. 2a). The reduction in steady-state level predominantly
affected the mature form generated by spliceosomal cleavage,
whereas the levels of precursor and spliced form were only
slightly reduced (Fig. 2a, b). The levels of Smb1, Sme1, Lsm4,
Lsm5 also remained unchanged in the absence of Pof8, and the
level of Trt1 protein was only slightly reduced indicating that the
reduction in the mature form of TER1 is a direct consequence of
loss of Pof8 protein (Supplementary Fig. 1). Consistent with the
observations of the genome-wide telomere length screen20, we
found telomere length to be very short in the absence of Pof8
(Fig. 2c). It is important to note that the chromosome terminal
fragments following digest of genomic DNA with EcoRI are
composed of ~800 bp subtelomeric DNA and a variable number
of telomeric repeats. The difference in mobility between wildtype
and pof8? is therefore indicative of critically short telomeres with
most of the remaining fragment composed of subtelomeric DNA.
Although telomeres were maintained at this short length over
successive restreaks at the population level, a fraction of telomeres
nevertheless became uncapped and chromosome end fusions
were readily detected in pof8? cells, but not in wild-type cells
under the same condition (Fig. 2d). A ter1? strain in crisis served
as a positive control for chromosome end fusions.
Pof8 deletion impairs telomerase activity. The pronounced
telomere defect observed here was difficult to reconcile with the
modest reduction in telomerase RNA level observed by northern
blotting. To investigate whether telomerase activity was affected
more severely than expected from the four-fold reduction in
TER1 RNA, we performed direct telomerase activity assays from
Trt1 immunoprecipitates prepared from cell extracts of pof8+ and
pof8? cells. We observed a 20- to 30-fold reduction in telomerase
activity in the absence of Pof8 (Fig. 2e). Even less telomerase
activity was detected when telomerase was immunoprecipated
with Lsm proteins from pof8? cells (Fig. 2f).
To separate the effects of pof8 deletion on TER1 stability from deletion strains (lanes 5 and 6) compared to wildtype (lane 1).
a role for Pof8 in telomerase biogenesis or regulation of activity, Regardless of the amount of TER1, telomerase activity was
we expressed TER1 from the inducible nmt1 promoter (Fig. 3a). dramatically reduced in the absence of Pof8 (Fig. 3b). Only after
In the induced state, TER1 levels were 10-fold higher in the pof8 contrast adjusting beyond the point of saturation for the signal
pof8 + pof8?
+ ? + ? RT
from pof8+ samples, weak telomerase activity was detected in the
pof8? extracts (Fig. 3b, lower panel). Despite higher levels of
TER1 RNA in the induced pof8? cells compared to the uninduced
pof8+ cells, the activity was over 300-fold reduced (compare lanes
2?4 with 14?16). Furthermore, despite a 16-fold higher level of
telomerase RNA in induced pof8? cells vs. uninduced cells, the
activity increased by less than two-fold. Consistent with the low
activity despite overexpression of TER1, the short telomere
phenotype of pof8? cells was not rescued (Fig. 3c). TER1 levels
also increased following deletion of the RNA exonuclease rrp6
(Supplementary Fig. 2a)23. In line with the results obtained by
overexpressing TER1, deletion of rrp6 also failed to rescue
telomerase activity (Supplementary Fig. 2b). In summary,
deletion of pof8 caused a four-fold reduction in the steady-state
level of TER1, but had a far more dramatic effect on telomerase
activity. Overexpressing TER1 or interfering with TER1
degradation rescued the RNA level, but failed to rescue telomerase
activity and telomere shortening.
Effect of pof8 deletion on telomerase assembly. The strong
effect of pof8 deletion on telomerase activity indicated that Pof8
may function as an assembly factor for telomerase. As shown
previously8, Lsm2-8 proteins are associated with the majority of
mature telomerase RNA, whereas Sm proteins are bound to the
precursor and a minor fraction of mature TER1. Deletion of pof8
results in a 20-fold reduction in the amount of TER1
immunoprecipitated with Lsm4 and Lsm5, and a slight increase in Smb1
pof8 + pof8?
8? A A B
8fop fop U I U I U I
and Sme1 association (Fig. 4a). These results indicate that loading
of Lsm proteins onto TER1 is compromised in the absence of
Pof8. To exclude the possibility that the reduction in TER1 level
caused by the deletion of pof8 was responsible for the diminished
recovery by immunoprecipitation, we repeated the experiment in
the context of overexpressed TER1 (Fig. 4b). Whereas
immunoprecipitation of Lsm4 from a pof8+ strain depleted over 50% of
TER1 from the supernatant (lane 3), no measurable depletion was
observed in the pof8? extract (lane 4, compare input with S/N)
and TER1 was barely detectable in the immunoprecipitate
(IP, lane 4, lower panel). We conclude that Lsm association with
TER1 is compromised in the absence of Pof8. As we have
previously shown that Trt1 association with TER1 requires prior
binding of Lsm2-88, these experiments place Pof8 upstream of
Lsm and Trt1 in the hierarchical assembly of telomerase and
explain the dramatic reduction in telomerase activity in the
absence of Pof8.
We next wanted to know whether Pof8 directly and stably
associates with TER1. Following introduction of an N-terminal
3xFLAG epitope tag, Pof8 was detected as a single band by
western blotting (Fig. 5a). Telomeres continued to be maintained
at near wildtype length indicating that the 3xFLAG tag has little
effect on Pof8 function (Fig. 5b). To test whether Pof8 binds
directly to TER1, we incubated a radiolabeled probe
corresponding to the short arms of TER1 with S. pombe extract containing
FLAG-tagged Pof8 and UV irradiated to crosslink RNA?protein
interactions. Pof8 was then immunoprecipitated under
denaturing conditions to disrupt indirect interactions. Parallel control
experiments were carried out with extracts containing no tags and
myc epitope-tagged Lsm4, respectively. RNA was isolated from
each IP, separated by gel electrophoresis and visualized (Fig. 5c).
TER1 was found to be 2.5-fold enriched in the Lsm4 and Pof8 IPs
relative to the untagged control, strongly supporting that Pof8
directly interacts with TER1, as does Lsm4.
Pof8 is a subunit of active telomerase. To assess whether
Pof8 stably associates with TER1 in the context of active enzyme,
we analyzed FLAG-Pof8 immunoprecipitations carried out under
native conditions by northern blotting. TER1 was readily detected
in immunoprecipitates from tagged, but not from control extracts
in which Pof8 was untagged (Fig. 6a). The snRNA snR101 was
not precipitated by Pof8 and thus served as specificity control.
Whereas no activity was observed in control samples, FLAG-Pof8
immunoprecipitates displayed robust telomerase activity
(Fig. 6b). To assess more quantitatively what fraction of
telomerase is Pof8-associated, we generated strains containing
Lsm4cMyc in combination with FLAG-tagged or untagged Pof8.
Cellfree extracts from these strains were subjected to a first round of
immunoprecipitation using anti-FLAG antibody. The
supernatant was then incubated with anti-cMyc to precipitate
Lsm4associated TER1. The four immunoprecipitates were then assayed
for telomerase activity. As expected, no telomerase activity was
precipitated in the FLAG IP from extract containing untagged
Pof8 (Fig. 6c, lanes 2?4). Subsequent IP of Lsm4-cMyc from the
supernatant of the first-round IP recovered robust activity (lanes
6?8). In contrast, when Pof8-associated telomerase was first
precipitated (lanes 10?12), only 1% of activity was recovered in a
subsequent Lsm4 IP (lanes 14?16). Based on these results, nearly
100% of active telomerase are associated with Pof8, making this
protein a bona fide component of the active holoenzyme.
Pof8 is not a general loading factor for Lsm2-8. Considering the
homology with La-related proteins and the stable association with
telomerase, we wondered whether other RNAs were also affected
by deletion of pof8. Using ribo-depleted RNA from otherwise
isogenic pof8+ and pof8? strains, we performed expression
analysis in triplicate. Five protein-encoding transcripts and 13
noncoding RNAs including TER1 were found to be expressed at more
than two-fold lower levels in pof8? cells (Supplementary
Table 3a). An even smaller number of transcripts was found to be
upregulated by greater than two-fold (Supplementary Table 3b).
Among the seven upregulated protein-encoding genes was tlh2, a
locus located in subtelomeric DNA and previously found to be
upregulated in cells with critically short telomeres24.
Although U6 snRNA associates with the Lsm2-8 complex like
TER1, the U6 expression level was unaffected by the presence or
absence of Pof8. Furthermore, immunoprecipitates from
FLAGPof8 extracts contained barely more U6 than control IPs from
extracts with untagged Pof8 (Fig. 7a). This argues against Pof8
being a general loading factor for Lsm2-8 and a component of the
U6 snRNP. To further assess the specificity of Pof8 for loading
the Lsm complex onto RNAs, we asked how many other RNAs
that are associated with Lsm8 are affected in abundance by
deletion of pof8. We chose Lsm8 for this experiment as it is the
only Lsm family member unique to the Lsm2-8 complex, all
others being shared by the cytoplasmic Lsm1-7 complex.
Immunoprecipitation of Lsm8-cMyc enriched 35 RNAs by
greater than two-fold, including U6 (30-fold enrichment) and
TER1 (159-fold enrichment) (Supplementary Table 4).
Overlaying the differential expression data for pof8 with the Lsm8 IP
revealed TER1 as the only transcript that is bound by Lsm8 and
reduced in pof8? cells (Fig. 7b).
In summary, our results demonstrate that Pof8 is a previously
unrecognized member of the LARP family that shares structural
and functional similarity with telomerase subunits from ciliated
protozoa, namely p43 and p65. Pof8 binds to telomerase RNA
and promotes the loading of the Lsm2-8 complex, which in turn
promotes the loading of the catalytic subunit Trt1. Pof8 is
associated with nearly all telomerase activity, establishing the protein
as a bona fide protein component of functional telomerase in
The identification of La-related proteins in association with
highly purified telomerase from Euplotes and Tetrahymena was
initially seen as unsurprising due to the nature of these RNAs as
pol III transcripts. La protein associates rapidly and in most cases
transiently with nascent pol III transcripts, and guides the RNAs
through various processing steps25. Interestingly though,
subsequent studies revealed that p43 and p65 are telomerase-specific
proteins, suggesting functions distinct from those provided by the
canonical La proteins15,17. A series of elegant biochemical15,26,
structural27,28, and single-molecule29 experiments have since
demonstrated that p65 binding to telomerase RNA induces
structural changes in the RNA that are instrumental for the
hierarchical assembly of functional telomerase in Tetrahymena.
Much less is known about the assembly pathways for
telomerases from other organisms, but recent findings have revealed
more differences than commonalities. Budding yeast TLC1 is
stably associated with Sm proteins9, whereas fission yeast TER1
undergoes the Sm to Lsm switch8. While divergent yeast
telomerase RNAs share features with snRNAs, vertebrate TRs belong
to the family of H/ACA box snoRNAs30 and scaRNAs31, and
associate with a different set of proteins to mediate 3? end
processing and stability32?35. The characterization of several
telomerase components that lack clear functional orthologs in other
eukaryotes supports the view that telomerases evolve far more
rapidly than other well-characterized RNPs, and that the presence
of a reverse transcriptase and a highly divergent non-coding RNA
may be the only common denominators. Along these lines, it was
recently shown that Pop1, 6, and 7, previously characterized as
binding the P3 domains of RNaseP and MRP, are also
constitutive components of telomerase in budding yeast and are
critical for holoenzyme integrity in this organism36,37. Whether
this remarkable feature of a conserved functional module being
shared between three highly divergent ancient RNPs is conserved
in other species remains to be seen. It appears that telomerase
RNA easily acquires functional sequence modules, such as the P3
or H/ACA domains, and that active telomerase can be produced
via diverse pathways.
The identification of a La-related protein as a constitutive
telomerase subunit in fission yeast presents a different
perspective. This is the first telomerase subunit since the cloning of the
catalytic subunits in the late 1990?s that is structurally and
functionally conserved between organisms as distant as ciliates
and yeasts. Despite a nearly 10-fold difference in size between
ciliate and yeast TRs, a La-related protein has now been found to
function in telomerase biogenesis in a seemingly conserved
manner. Notably, two human La family members, LARP3 and
LARP7, have also been implicated in telomere maintenance.
LARP7 has been reported to affect alternative splicing of Tert38,
whereas LARP3 was found to associate with telomerase RNA39.
Whether either of these proteins functions in a similar manner to
p65 and Pof8 to promote telomerase assembly and activity
remains to be tested. Studies of p65 were instrumental in
establishing the paradigm of hierarchical assembly of functional
telomerase aided by RNA chaperones40. In S. pombe, Sm proteins
assemble on the precursor of TER1, promote spliceosomal
cleavage, and recruit Tgs1, which hypermethylates the mono-methyl
guanosine cap. Through the present analysis, a picture of an
ordered sequence of events emerges: Pof8 must bind to TER1
either before or immediately after spliceosomal cleavage, possibly
triggering the departure of Sm proteins. Pof8 binding is then a
prerequisite for Lsm2-8 loading onto the 3? end of TER1. This
may involve the recruitment of additional factors and/or a
Pof8induced conformational change in the RNA that creates a
highaffinity Lsm-binding site. Pof8 remains associated with the active
holoenzyme even after Lsm2-8, and subsequently Trt1, bind to
TER1. The requirement for Pof8 for the Lsm?TER1 association
and the formation of a Pof8?Lsm?Trt1?TER1 complex now
suggests that hierarchical assembly is a conserved path for
telomerase biogenesis that may also have a place in telomerase
assembly in metazoans.
Strains and constructs. S. pombe strains used in this study are listed in
Supplementary Table 1. The pof8 deletion was generated by replacing the complete open
reading frame with the kanamycin resistance cassette using standard laboratory
techniques41,42. Knockout fragments contained ~750 base pair (bp) upstream and
downstream homology, and were generated by fusion PCR using primers listed in
Supplementary Table 2. Cells were grown to late log phase and transformed by
lithium acetate method. Transformants were selected on YEA plates plus geneticin
disulfate (100 ?g ml?1). Epitope tags were introduced following the same strategy.
Other strains were generated by crossing and selection of correct genotypes. The
3xFLAG tag was introduced at the N-terminus of Pof8 by fusion PCR in the
context of a genomic fragment encompassing sequence from position ?386 to
+1769 and cloned into pDBlet plasmid43. All strains were verified by PCR or
western blotting. Plasmids were introduced into S. pombe cells by electroporation.
Telomere length analysis and fusion assay. DNA preparation and telomere
length analysis were performed based on ref. 44. Cells from 20 ml cultures (~1 ? 109
cells per ml) were incubated with 2 ml of Z buffer (50 mM sodium citrate, 50 mM
sodium phosphate dibasic, and 40 mM EDTA pH 7.8) plus 0.5 mg ml?1 Zymolase
T100 (US Biological) and 2 mM dithiothreitol (DTT) for 1 h at 37 ?C. Sodium
FLAG IP cMyc IP
FLAG IP cMyc IP
Myc Myc Beads
Myc Myc Beads
100-mer oligonucleotide was used as LC. c Telomerase assay of samples generated by sequential IPs with FLAG and cMyc antibodies. A schematic of the
experiment is shown above the blot: first, extracts were exposed to ?-FLAG antibody-bound beads, the supernatant (S/N) from the ?-FLAG IP was then
exposed to ?-cMyc antibody-bound beads followed by a telomerase assay for all groups of IP samples. The uncropped blot is presented in Supplementary
dodecyl sulfate (SDS) was then added to a final concentration of 2% (w/v) and
incubated for 10 min at 65 ?C. Then 5? TE (50 mM Tris-HCl pH 8.0, 5 mM EDTA)
was added to a final volume of 10 ml and proteinase K (Sigma-Aldrich, P2308) to a
final concentration of 50 ?g ml?1. After incubation for 1 h at 50 ?C, the samples
were precipitated with 3 ml of 5 M potassium acetate for 30 min on ice. The
precipitates were removed with two rounds of centrifugation at 3200 ? g for 10 min at
4 ?C. The supernatant was collected and mixed with 1 volume of 100% isopropanol
for 1 h on ice followed by centrifugation at 10,500 ? g for 10 min at 4 ?C. Genomic
DNA was resuspended in 5? TE with 50 ?g ml?1 RNAse A. Resuspended DNA was
then incubated for 1 h at 37 ?C followed by two rounds of extraction with phenol:
chloroform:isoamyl alcohol (25:24:1, equilibrated with 5? TE) and one round of
chloroform:isoamyl alcohol (24:1, equilibrated with 5? TE). DNA was ethanol
precipitated and resuspended in 1? TE. DNA concentrations were determined on a
Qubit 3.0 instrument using the dsDNA BR Assay Kit (Life Technologies, Q32853)
and 750 ng of each sample was digested with EcoRI for 12 h and then loaded onto a
1% agarose gel. The digested DNA was electrophoresed in 0.5? TBE (44.5 mM
Tris-borate, 1 mM EDTA at pH 8.3) at 120 V for 6 h. Gels were stained with 1 ?g
ml?1 ethidium bromide and visualized with Typhoon 8600 scanner to confirm
digestion of loaded DNA. Gels were then incubated in 0.25 M hydrochloric acid for
10 min followed by 0.5 M sodium hydroxide and 1.5 M sodium chloride buffer for
30 min and 0.5 M Tris-HCl (pH 7.5) and 1.5 M sodium chloride for 30 min at
room temperature. DNA was transferred to Amersham Hybond-N+ membrane
(GE Healthcare Life Sciences) via capillary blotting. Transferred DNA was
crosslinked to the membrane in a Stratalinker using a 254-nm UV light at 120 mJ cm?2.
A probe specific for telomeric sequences was generated by PCR from pTELO using
T3 (5?-ATTAACCCTCACTAAAGGGA-3?) and T7
(5?-TAATACGACTCACTATAGGG-3?) oligos. A probe specific for the rad16 gene was generated by PCR from
wild-type genomic DNA using primers XWP9 (5?-ATGGTATTTTTTCGCCATT
TACTCG-3?) and XWP10 (5?-TAGGCGGATCGTGAAGTTAA-3?). Both probes
were labeled by random hexamer labeling with High Prime (Roche, 11585592001)
and [?-32P]-dCTP. Hybridizations were carried out with 10 million counts per
minute of probe in Church?Gilbert buffer45 at 65 ?C. Blots were exposed to
PhosphorImager screens and visualized with a Typhoon 8600 scanner.
To amplify chromosome end fusions, PCR reactions (25 ?l) contained 1?
ThermoPol buffer (NEB), 200 ?M dNTPs, 0.5 ?M of Bloli1256 (GGGTTGCAAA
GTATGATTGTGGTAA), and Bloli1353 (TGTTGAATGTCAGAACCAACTGTT
Input S/N IP
GCAT) to amplify fusion junctions, 0.1 ?M of Bloli3400 (GCAAAGAAGTTTCC
TGGAATAGC) and Bloli3405 (GATGTAATAAAGGGTCGGCAC) to amplify
part of the trt1 gene as loading control, 1.25U Taq polymerase (NEB, M0273) and
1 ng of genomic DNA. Reactions were incubated at 95 ?C for 30 s, followed by 32
cycles of 95 ?C for 15 s, 55 ?C for 30 s, and 68 ?C for 3 min with a final extension at
68 ?C for 10 min.
Native protein extract and immunoprecipitation. Cultures (2 l) were grown to a
density of 0.5?1 ? 107 cells per ml and harvested by centrifugation for the
preparation of cell-free extract3,44. Cells were washed three times with ice-cold TMG
(300) buffer (10 mM Tris-HCl pH 8.0, 1 mM magnesium chloride, 10% (v/v)
glycerol, 300 mM sodium acetate), and resuspended in two packed cell volumes of
TMG(300) plus complete EDTA-free protease inhibitor cocktail (Roche), 0.5 mM
PMSF, 1 mM EDTA, and 0.1 mM DTT and quick-frozen by dripping the cells
suspension in small droplets into liquid nitrogen. Cells were lysed in a 6850 Freezer
mill (SPEX SamplePrep) using eight cycles (2 min) at a rate of 10 per second with
2 min cooling time between cycles. Lysates were thawed on ice and one additional
packed cell volume of TMG(300) plus supplements was added. Lysates were then
cleared by centrifugation twice for 10 min at 6000 ? g in a Beckman JA-17 rotor
and then once for 45 min in a Beckman 70Ti rotor at 36,000 ? g. All steps were
carried out at 4 ?C. Protein concentration was determined by Bradford assay and
ranged between 6 and 11 mg ml?1.
For RNA IPs, extracts (5.5 mg) were diluted to 5 mg ml?1 with TMG(300)
buffer plus supplements. An aliquot (100 ?l) was frozen as input control. Heparin
was added to 1 mg ml?1 and Tween-20 to 0.1% (v/v). Magnetic dynabeads protein
G (30 mg ml?1; Invitrogen) was coated with anti-c-Myc 9E10 or anti-FLAG M2
(10 ?g per 50 ?l of bead suspension; Sigma-Aldrich, M4439 and F3165) by
incubation for 30 min at room temperature in 200 ?l of 1? PBS + 0.1% (v/v)
Tween-20. Beads were washed three times with 1 ml of TMG(300).
Immunoprecipitation was performed with 60 ?l (Pof8) and 120 ?l (Sm/Lsm) of
bead suspension for 4 h at 4 ?C with gentle rotation. Beads were collected using a
magnet and an aliquot (100 ?l) of supernatant was removed and frozen for further
analysis. The beads were then washed five times with 1 ml TMG(300) plus
supplements and 0.1% (v/v) Tween-20, once with TMG(200) (as TMG(300) except
sodium acetate was at 200 mM) plus supplements and 0.1% (v/v) Tween-20 and
once with TMG(50) plus supplements. Finally, beads were resuspended in 120 ?l
TMG(50) plus supplements and 0.4 U ?l?1 RNAsin (Promega) and frozen in liquid
RNA preparation. For total RNA extraction, cells (500 ml) were grown to a density
of 5 ? 106 cells per ml and collected by centrifugation, washed twice with ddH2O
(500 ml), resuspended in 3 ml ddH2O and quick-frozen by dripping the cells
suspension in small droplets into liquid nitrogen. Cells were lysed in 6850 Freezer
mill (SPEX SamplePrep) using seven cycles (2 min) at a rate of 10 per second with
2 min cooling time between cycles. The lysed cells were transferred into 50 ml tubes
containing 10 ml phenol:chloroform:isoamyl alcohol (25:24:1, equilibrated with
50 mM sodium acetate, pH 5.2) and 10 ml 50 mM sodium acetate and 1% (w/v)
SDS preheated to 65 ?C. RNA was extracted four times with 10 ml phenol:
chloroform:isoamyl alcohol (25:24:1, equilibrated with 50 mM sodium acetate, pH
5.2) and once with chloroform:isoamyl alcohol (24:1, equilibrated with 50 mM
sodium acetate, pH 5.2). Total RNA was ethanol precipitated and resuspended in
In the context of immunoprecipitation experiments, RNA was isolated from
input, supernatant, and beads by incubation with proteinase K (2 ?g ?l?1 in 0.5%
(w/v) SDS, 10 mM EDTA pH 8.3, 20 mM Tris-HCl pH 7.5) at 50 ?C for 15 min,
followed by extraction with phenol:chloroform:isoamyl alcohol and chloroform:
isoamyl alcohol. RNA was ethanol precipitated for 4 h at ?20 ?C and resuspended
in ddH2O. RNA used for RT-PCR was further DNase treated using the RNeasy
Mini Kit (Qiagen) following the manufacturer?s instructions.
Northern blot analysis. Where indicated, RNaseH cleavage was carried out on 15
?g of DNAse-treated total RNA isolated from S. pombe. RNA was combined with
600 pmol of BLoli1043 (5?-AGGCAGAAGACTCACGTACACTGAC-3?) and
BLoli1275 (5?-CGGAAACGGAATTCAGCATGT-3?) targeting exon 1 and exon 2,
respectively. The mixture was heated to 65 ?C in a thermocycler for 5 min and then
allowed to slowly cool down at room temperature for 10 min. About 1? RNaseH
buffer (NEB) and 5U RNaseH enzyme (NEB, M0297) were added to the mixture
and incubated for 30 min at 37 ?C. RNaseH-treated samples were ethanol
precipitated for 4 h at ?20 ?C and centrifuged at 2000 ? g for 20 min at 4 ?C. RNA was
then resuspended in 1? formamide loading buffer and separated on a 4% (v/v)
polyacrylamide (29:1) gel containing 8 M urea and transferred to Biodyne nylon
membrane (Pall Corporation) at 400 mA for 1 h in 0.5? TBE buffer. RNA was
crosslinked to the membrane using 254-nm UV light at 120 mJ cm?2 in Stratalinker
(Stratagene). Hybridization with radiolabeled probes (10 million counts per
minute) were performed in Church?Gilbert buffer45 at 60 ?C for TER1 probe
(nucleotides 536?998, labeled with High Prime (Roche) and [?-32P]-dCTP), and at
42 ?C for small nucleolar RNA snRN101 and U6 snRNA (oligonucleotide
BLoli1136 (5?-CGCTATTGTATGGGGCCTTTAGATTCTTA-3?) and BLoli4628
(5?-TCTGTATCGTTTCAATTTGACCAAAGTGAT-3?), respectively, labeled with
T4 polynucleotide kinase (NEB, M0201) in the presence of [?-32P]-ATP). Blots
were exposed to PhosphorImager screens and analyzed with a Typhoon
8600 scanner. The uncropped blots of this work are shown in Supplementary Fig. 3.
Western blot analysis. Western blot analysis was performed with native protein
extracts, prepared as described above, diluted to 6 ?g ?l?1 and mixed with equal
volume of 2? protein sample buffer (2? NuPAGE LDS buffer (Life Technologies),
100 mM DTT, 4% (w/v) SDS). Samples were then incubated for 10 min at 75 ?C
and 10 ?l (30 ?g) of samples was loaded onto a 4?12% NuPAGE Bis-Tris gel (Life
Technologies, NP0321BOX). Electrophoresis was done in 1? MOPS buffer (Life
technologies, NP0001) at 160 V for 60 min. Proteins were transferred to Protran
nitrocellulose membranes (Whatman) in western transfer buffer (3.03 g l?1 Tris
base, 14.4 g l?1 glycine, 20% (v/v) methanol) at 100 V for 1 h. Blots were blocked
and washed with iBind Flex Western Device (Life Technologies, SLF20002). Lsm
and Sm blots were probed with mouse monoclonal anti-c-Myc 9E10
(SigmaAldrich, M4439) at 1:5000 dilution and horse-radish peroxidase-conjugated goat
anti-mouse IgG (H+L) at 1:5000 (Thermo Scientific, 31430). Trt1 blot was probed
with rabbit polyclonal anti-cMyc A14 (Santa Cruz Biotechnologies, sc-789) at 1:400
dilution and horse-radish peroxidase-conjugated goat anti-rabbit IgG (H+L) at
1:4000 (Thermo Scientific, 31460). Blots were reprobed with mouse anti-?-tubulin
RT-PCR. DNAse-treated RNA samples were used for RT-PCR reaction as
describe8. Primers for RT reaction were BLoli1275
(5?-CGGAAACGGAATTCAGCATGT-3?) for precursor and spliced form; PBoli918
(5?ACAACGGACGAGCTACACTC-3?) for first exon; and BLoli2051 (5?-GACCT
TAGCCAGTCCACAGTTA-3?) for U1 as loading control. RNA samples (2.5 ?g)
were combined with oligos (10 pmol) and dNTP mix (10 nmol) in 13 ?l, and
samples were heated to 65 ?C for 5 min. After cooling, the volume was increased to
20 ?l by the addition of 40 U of RNasin (Promega), 5 mM DTT, 1? first-strand
buffer, and 200 U of Superscript III reverse transcriptase (Invitrogen). Samples
were incubated at 55 ?C for 60 min. RNaseH (5 U, NEB, M0297S) was added
followed by incubation at 37 ?C for 20 min. Aliquots (2 ?l) of the RT reactions were
used for PCR amplification with Taq polymerase (NEB, M0273) under the
following conditions: 5 min at 94 ?C followed by 28 cycles of 30 s at 94 ?C, 30 s at 57 ?
C, and 60 s at 72 ?C, followed by 10 min at 72 ?C. Primers used were BLoli1275 and
Bloli1020 (5?-CAAACAATAATGAACGTCCTG-3?) for precursor and spliced
form, PBoli918 and BLoli1006 (5?-CATTTAAGTGCTTGTCAGATCACAACG-3?)
for first exon, and BLoli2051 and BLoli2101 (5?-ACCTGGCATGAGTTTCTGC-3?)
Telomerase activity assay. Telomerase was immunoprecipitated on magnetic
dynabeads protein G (Invitrogen, 10003D) coated with anti-c-Myc 9E10
(SigmaAldrich, M4439) for Trt1 and LSm4, LSm5 or anti-FLAG M2 (Sigma-Aldrich,
F3165) for Pof8 as described above. Three amounts of bead suspension (5, 10, and
20 ?l) were used for the telomerase activity assay. Negative control samples were
incubated in 20 ?l of TMG(50) plus 20 ng of RNAse A (Invitrogen) for 10 min at
30 ?C. Beads were incubated in 10 ?l of 50 mM Tris-acetate at pH 8.0, 100 mM
potassium acetate, 1 mM magnesium acetate, 5% (v/v) glycerol, 1 mM spermidine,
1 mM DTT, 0.2 mM dATP, dCTP, dTTP, 2 ?M [?-32P]-dGTP (500 Ci mmol?1),
and 5 ?M of oligo PBoli871 (5?-GTTACGGTTACAGGTTACG-3?). Reactions were
incubated for 90 min at 30 ?C and stopped by the addition of proteinase K (2 ?g ?l
?1 in 0.5% (w/v) SDS, 10 mM EDTA pH 8.3, 20 mM Tris-HCl pH 7.5) plus
1000 cpm 100-mer labeled with [?-32P]-ATP as the loading control at 42 ?C for
15 min. Primer extended products were extracted with phenol:chloroform:isoamyl
alcohol (25:24:1, equilibrated with 5? TE) and ethanol precipitated for 4 h at ?20 ?
C. Extracted DNA was electrophoresed in 10% (v/v) polyacrylamide (19:1)
sequencing gel containing 8 M urea for 1.5 h at 80 W. Gels were dried and exposed
to PhosphorImager screens, and analyzed with a Typhoon 8600 scanner.
UV crosslinking and denaturing immunoprecipitation. A Ter1 probe corre
sponding to the two short arms was generated by fusing an Sp6 promoter sequence
with nucleotides +1 to +97 and +955 to +1212 of TER1 and a hepatitis ? virus
(HDV) ribozyme sequence to allow for production of a precisely defined 3? end.
Primers used to generate the DNA template are listed in Supplementary Table 2.
The TER1 +1 to +97 fragment with Sp6 promoter sequence was amplified from
pJW103 with BLoli7098 (containing Sp6 promoter sequence) and BLoli7099
(containing 20 nt overlapping sequence with +955 to +1212 fragment). The TER1
+955 to +1212 fragment with HDV sequence (5?-GGGCGGCATGGTCCCAGC
CCAA-3?) was PCR amplified from pTER1-i33 with primers Bloli7100 and
Bloli6540. The entire probe sequence was then amplified by fusion PCR from the
two fragments as template. PCR reactions (50 ?l) contained 1? Phusion HF buffer
(Life Technologies), 200 ?M dNTPs, 0.5 ?M of each primer, and 0.02 U ?l?1 of
Phusion Hot Start II DNA Polymerase (Life Technologies, F549). Reaction
conditions were: 98 ?C for 30 s, followed by 32 cycles of 98 ?C for 10 s, 65 ?C for
30 s, and 72 ?C for 30 s with a final extension at 72 ?C for 10 min. The PCR product
was cloned using Zero Blunt PCR Cloning Kit (Life Technologies, K270020)
following manufacturer?s instructions to give rise to plasmid pDP2 which was
sequence verified. The template for in vitro transcription was generated by PCR
amplification from plasmid pDP2 with primers BLoli7098 and BLoli6540 using the
PCR conditions listed above.
The in vitro transcription reactions contained 1? transcription buffer
(Promega), 0.5 mM each of ATP, CTP, GTP, 0.1 mM UTP, 0.66 ?M [?-32P]-UTP
(3000 Ci mmol?1), 1 ?g DNA template, 40 U RNasin (Promega, N2111), and 1.9 U
SP6 RNA polymerase (Promega, P1085) in a 10 ?l volume. Reactions were
incubated at 37 ?C for 3 h (time optimized to maximize the amount HDV ribozyme
cleaved product), and 2 U of DNAse I (NEB, M0303) was added, followed by
incubation for 30 min at 37 ?C. Reactions were stopped by the addition of
formamide loading dye. The full-length HDV-cleaved TER1 probe product was gel
purified on 5% (v/v) polyacrylamide gels containing 8 M urea for 2 h at 15 W. The
transcribed and processed RNA is predicted to fold into the same structure as in
the context of the full-length RNA using mFOLD default parameters (http://
To capture proteins that directly interact with TER1, the RNA probe (2.0 nM)
was incubated on ice for 30 min with native protein extracts from strains
containing cMyc-tagged Lsm4, FLAG-tagged Pof8, and no tag, respectively.
Reactions (100 ?l) contained 1.5% (w/v) PEG8000 (NEB), 60 mM potassium
phosphate pH 7.0, 1 mM spermidine, 2 mM MgCl2, 0.4 U of RNAsin (Promega,
N2111), and 40 ?l of 8 ?g ?l?1 native protein extract. After incubation, 50 ?l of the
reaction was aliquoted into two 25 ?l droplets onto parafilm stretched over an
aluminum block that was precooled at 4 ?C and irradiated in a Stratalinker
(Stratagene) using 254 nm UV light at 0.8 J cm?2. After crosslinking, 1% (w/v) of
SDS, 1% (v/v) of Triton X-100, and 100 mM DTT were added to the crosslink and
no crosslink reactions, and then heated in boiling water for 2 min. Denatured
samples were diluted 10-fold with TMG(300) buffer and immunoprecipitated with
magnetic dynabeads protein G (Invitrogen) coated with anti-c-Myc 9E10 or
antiFLAG M2 (Sigma-Aldrich, M4439 and F3165) antibody as described in native
protein extract and immunoprecipitation section. After the immunoprecipitation,
beads were washed four times with 1 ml TMG(300) and once with 1 ml TMG(50),
and treated with proteinase K (2 ?g ?l?1 in 0.5% (w/v) SDS, 10 mM EDTA pH 8.3,
20 mM Tris-HCl pH 7.5) at 42 ?C for 15 min. The supernatant was extracted with
phenol:chloroform:isoamyl alcohol (25:24:1, equilibrated with 50 mM sodium
acetate, pH 5.2) and ethanol precipitated for 4 h at ?20 ?C. RNA was resolved on a
5% (v/v) polyacrylamide containing 8 M urea for 2 h at 15 W. Gels were dried onto
Whatman 3MM Chr blotting paper (GE Healthcare Life Sciences) and exposed to a
PhosphorImager screen for analysis with a Typhoon 8600 scanner.
RNA-Seq and RIP-Seq. Ribo-depleted stranded RNA-Seq libraries were
constructed for three Pof8 samples and isogenic controls using the TruSeq SBS Kit
v4HS kit (Illumina). The libraries were pooled and sequenced on an Illumina HiSeq
2500 for 100-bp single-end reads on two lanes. Read counts per library ranged
from 56 million to 84 million. Reads were aligned to the S. pombe ASM294v2
genome from ensembl using the STAR aligner46 (v.2.5.2b) with the following
parameters: -outFilterType BySJout --alignSJDBoverhangMin 5
--alignSJoverhangMin 10 --alignIntronMin 20 --alignIntronMax 2500 --twopassMode Basic.
Between 49 and 72 million reads per library passed filtering. A counts table for
unambiguous uniquely mapped reads was generated using a custom R script.
Coordinates for snu4 (II: 467,488?467,615)47 and snu5 (II: 3,236,356?3,237,312)
(GenBank: X16573.1) were manually curated based on alignment of the published
sequences to the genome. Differential gene expression analysis was performed with
EdgeR48 (v.3.14.0) using the likelihood ratio test with the Benjamini?Hochberg
false discovery rate correction. Genes with fewer than one count per million in
three or more libraries were filtered prior to differential expression analysis. Genes
with absolute log2 fold change >1 with an adjusted p-value < 0.05 were considered
RNA libraries from Lsm8 and control immunoprecipitations were prepared
following instructions for Solexa messenger RNA sequencing and each sample was
sequenced on a single lane of the Illumina GAIIx sequencer producing 40 bp
single-end unstranded reads. The fastq files were aligned to the S. pombe reference
genome ASM294v2 using the STAR aligner (v 2.5.2b) with the following
parameters: --outFilterType BySJout --outFilterMultimapNmax 20
--alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --alignIntronMin 20
--alignIntronMax 1000 --outSAMtype BAM SortedByCoordinate --twopassMode
Basic. The corresponding reference annotation gtf file was manually curated for
snu4 using coordinates II:467,488?467,615. A counts table for unambiguous
uniquely mapped reads was generated using a custom R script. There were 942,516
read counts for the control IP and 6,604,836 reads for the Lsm8 IP sample. The
read counts for each gene were normalized to the median read counts per library.
Genes were called enriched if the normalized counts in the IP sample exceeded the
corresponding counts in the control by 10 or more and the normalized read count
ratio was >2.
Data availability. Original data underlying this manuscript can be accessed from
the Stowers Original Data Repository at http://www.stowers.org/research/
publications/LIBPB-1190. Gene expression data and immunoprecipitation data
have been deposited in NCBI?s Gene Expression Omnibus (GEO) database under
accession number GSE104672.
We thank Li Chen for plasmid pTER1-i33, Katie Hildebrand for technical assistance,
Kristi Jensen and Lisa Lassise for help with preparing the manuscript, and Chi-Kang
Tseng and other members of the Baumann laboratory for discussions. We also thank
Laura Collopy, Kazu Tomita, and Toru Nakamura for sharing data prior to publication.
This work was funded in part by the Stowers Institute for Medical Research and was
performed in part to fulfill, in part, requirements for D.J.P.-M.?s thesis research in The
Graduate School of the Stowers Institute for Medical Research. P.B. is an Investigator
with the Howard Hughes Medical Institute.
D.J.P.-M. and P.B. designed the study, D.J.P.-M. carried out most of the experiments
except for data presented in Figs. 2d and 3c (L.P.) and part of Fig. 6b (W.T.). R.F.S.
processed the pof8 expression data and M.R.S. processed the RNA IP data set. D.J.P.-M.
and P.B. analyzed the data and prepared the manuscript.
Supplementary Information accompanies this paper at
Competing interests: The authors declare no competing financial interests.
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