Supplemental figure legends
Fig. S1: Accumulation of P-A3, P’-A3 and 18S-A3 fragments in rrp4-KD lines.
Northern analysis. Total RNA isolated from leaves of WT and mutant plants was separated
with extended electrophoresis time to achieve resolution of P’-A3 (detected with probe S2)
and 18S-A3 fragments (detected with both S2 and S4). The detected precursor species as
drawn in Fig. 1b are labelled on the right. The two bands migrating above the P-3’ ETS
precursor are the primary rRNA transcript (**) and the first precursor trimmed from the 5’
end prior to cleavage at the P-site (*).
Fig. S2: RRP6L2 interacts with RRP47 via the PMC2NT domain.
(a) Yeast two-hybrid assay showing that only RRP6L2 but not RRP6L1 or RRP6L3 can
interact with the RNA binding protein RRP47. The respective genotype of yeast
transformants is indicated in boxes representing each panel. AD47: RRP47 fused to the
activation domain of Gal4 (AD); BD6L1, BD6L2 and BD6L3: RRP6L1, L2 or L3 fused to
the DNA binding domain of Gal4 (BD), respectively. BD47, ADL1, ADL2 and ADL3
correspond to reciprocal fusions. Each strain was tested in triplicates with two dilutions for
growth on SD medium (-W-L-A) lacking Tryptophan (pGBDKT7 selection), Leucine
(pGADT7 selection) and Adenine (to select for bait-to-prey interaction). Growth tests on SDW-L-A medium revealed that only interaction of RRP47 with RRP6L2 provides adenine
auxotrophy. Growth tests on -W-L show that all yeast transformants grow under conditions
without selection for protein-protein interaction. All fusion proteins were detected by western
blot analysis (data not shown).
(b) Diagram of domains in the RRP6L2 protein. The PMC2NT domain is also present in yeast
and human RRP6 proteins, but not in Arabidopsis RRP6L1 or RRP6L3 (Lange et al. 2008).
The HRDC and EXO domains constitute the exoribonuclease domain. The bars below the
diagram show the constructs used for two-hybrid assays and pull-down experiments: (P)
PMC2NT region, (E) exoribonuclease domain, (C) C-terminal region of RRP6L2.
(c) and (d): The PMC2NT domain is sufficient for interaction with RRP47. RRP47 was
cloned into the pGBKT7 vector to fuse to the binding domain of Gal4. Constructs P, E or C
were cloned into the pGADT7 vector to fuse with the activation domain of Gal4 as indicated
on the right.
(c) Only coexpression of pGBKT7 RRP47 and pGADT7-P promoted growth in the absence
of Adenine.
(d) Pull-down experiments. In vitro translated and [35S]-methionine labelled proteins
corresponding to the PMC2NT region (P), the exoribonuclease domain (E) and the C-terminal
region (C) of RRP6L2 were mixed with RRP47 fused to a myc epitope in the presence or
absence of anti-myc monoclonal antibodies as indicated on the top. Input (10%) and eluates
were separated on SDS-PAGE before autoradiography. Only the PMC2NT region (P) coimmunoprecipitates with myc-RRP47.
Fig. S3: The PMC2NT domain of RRP6L2 is dispensable for degradation of 5'18S-A2
precursors.
Northern analysis of 18S precursors in different rrp6l2 alleles. While rrp6l2-1 and rrp6l2-3
are null alleles, a truncated RRP6L2 protein lacking the PMC2NT domain is produced in
rrp6l2-2 (Lange et al., 2008). The 18S-A2 precursor is detected in rrp6l2-1 and rrp6l2-3 but
not in rrp6l2-2, indicating that the PMC2NT is dispensable for its degradation (top panel). By
contrast, both rrp6l2-1 and rrp6l2-2 mutants accumulate similar amounts of 5.8S precursors
indicating that the low levels of truncated RRP6L2 produced in rrp6l2-2 mutants (Lange et al.
2008) are not sufficient for fully efficient maturation of 5.8S rRNA. Total RNA was separated
on 1.5% agarose gels (top) or 6% polyacrylamide/Urea gels (bottom) and hybridized with
probes S1-S5 as indicated on the left of each panel and in the diagram below the blots. The
detected precursors and degradation intermediates are indicated on the right. Methylene blue
staining (MB) was used as a loading control for the agarose gel. The 5S rRNA detected with a
specific oligoprobe is shown as a loading control for the polyacrylamide gel. fry1 represents
the fry1-6 mutant, where enzymatic activity of the 5'-3' exoribonucleases XRN2, XRN3 and
XRN4 is metabolically inhibited. Therefore, fry1-6 plants behave like a triple xrn2 xrn3 xrn4
mutant (Gy et al., 2007; Zakrzewska-Placzek et al., 2010). The trl mutant is described in this
study, see also Fig. S7. For each probe all samples were analysed on a single gel. White lines
indicate that lanes with additional samples were removed and are not shown in the figure.
Fig. S4: Loss of individual exosome components or cofactors does not result in the
accumulation of A2-A3 fragments.
Total RNA was separated on denaturing 6% polyacrylamide gels and hybridized with probe
S4 located in the ITS1 as indicated below the blot, 5S rRNA detected with a specific
oligoprobe was used a loading control. Since the accumulation of A2-A3 fragments in the
xrn2 xrn3 mutant was previously demonstrated (Zakrzewska-Placzek et al., 2010), the fry1
mutant was included as a positive control (see also legend of Fig. S3).
Fig. S5: Sequences of the 18S 3' RACE products shown in Fig. 3B.
The sequence of the ITS1 region located directly downstream of the mature 18S 3' end is
shown on the top. The forward primer used for the 3' RACE is indicated in yellow. The A2
processing site is marked in blue. PCR products cloned from WT, rrp6l1, rrp41 iRNAi,
rrp6l2 and rrp6l1 rrp6l2 samples are listed below. Non-encoded nucleotides are coloured in
blue (A), red (T) or green (G or C).
Fig. S6: Uridylation of pre-5.8S species.
3' RACE products were amplified as described in the legend of Fig. 5 except that o4 was used
as a forward primer. Vertical arrows indicate the location of the 3' ends of clones obtained
from WT, rrp41 iRNAi and rrp6l2 samples in the sequence of 5.8S precursors, together with
the length and the composition of non-encoded tails. The number of identical clones is given
in parentheses. Non-encoded nucleotides are coloured in blue (A), red (T) or green (G or C).
The diagram at the bottom illustrates the location of PCR primer with respect to the 5.8S
rRNA and the C2 processing site.
Fig. S7: TRL is a predominantly nucleolar homologue of yeast and human TRF4.
(a) Phylogenetic tree of the ten terminal nucleotidyl transferases that have been identified in
Arabidopsis, Saccharomyces cerevisiae Trf4 and Trf5, and human TRF4-2/PAD5. Sequences
corresponding to the nucleotidyl transferase and PAP/OAS1 substrate-binding domains of the
ten putative Arabidopsis TNTs, yeast Trf4 and Trf5, and human TRF4-2 were aligned with
ClustalW using the Phylogeny.fr platform and the PhyML program (Anisimova and Gascuel,
2006; Dereeper et al., 2008; Guindon and Gascuel, 2003). Graphical representation and
edition of the phylogenetic tree were performed with TreeDyn (Chevenet et al., 2006). (b)
Confocal microscopy of tobacco BY2 cells transiently expressing TRL fused to GFP (top)
and of Nicotiana benthamiana leaves co-expressing TRL-GFP and the nucleolar marker
protein Fibrilarin-RFP. DIC, differential interference contrast. Np: nucleoplasm; No:
nucleolus. (c) Diagram showing the location of T-DNA insertions present in trl-1 and trl-2
mutants with respect to the At5g53770 locus, exons are indicated by rectangles. Numbers
indicate the positions of start codon, stop codon and the left borders of the T-DNA insertions.
(d) Northern analysis showing reduced expression of TRL mRNA in trl-1 and trl-2 mutants.
MB, methylene blue.
Fig. S8: Loss of TRL reduces adenylation of pre-5.8 cleaved at C2.
3' RACE PCR on oligo-dT primed cDNA. A portion of the genomic sequence of the ITS2
region (starting 40 nt downstream of the mature 3' end of the 5.8S rRNA) is shown at the top.
The forward primer is indicated as horizontal arrow. The C2 processing site is marked by a
red arrow. The 3' ends of the PCR products that were cloned from WT, rrp41 iRNAi, trl-1
and trl-2 samples and corresponded to the ITS2 are listed below. Non-encoded nucleotides are
coloured in blue (A), red (T) or green (G or C). The numbers in parentheses indicate the
number of identical clones.
Fig. S9: Loss of TRL reduces adenylation of pre-18S cleaved at A3.
3' RACE PCR on oligo-dT primed cDNA. 3' ends of 18S precursors were amplified with
primer o3 located in the ITS1 as indicated in the diagram at the bottom. 24 clones obtained
from each WT, trl and rrp41 iRNAi samples were sequenced. Only clones that corresponded
to the target sequence are shown. Vertical arrows show the location of the polyadenylation
site in the ITS1 sequence. For each location, the composition of non-encoded oligonucleotide
tails is shown. The numbers in parentheses indicate the number of identical clones. Nonencoded nucleotides are coloured in blue (A), red (T) or green (G or C).
Fig. S10: Detection of polyadenylated P-P' fragments.
3' RACE-PCR on oligo-dT primed cDNA was performed with primer o1 located in the 5' ETS
as indicated in the diagram on the top. PCR products were separated in 2.5% agarose gels and
visualised with ethidium bromide staining. A size marker is shown on the right. All samples
were analysed in parallel. White lines indicate that lanes with additional samples were
removed and are not shown in the figure.
Fig. S11: Distinct degradation intermediates of the 5' ETS are detected in trl mutants.
3' RACE-PCR analysis of 3' extremities of 5' ETS fragments. Following 3' ligation of an RNA
adapter, cDNA synthesis was initiated with a primer complementary to the ligated adapter. 3'
RACE-PCR was performed with primer o1 and a primer complementary to the adapter. The
diagram on the bottom shows the location of the primer relative to the 18S rRNA and the
ITS1. The three panels above the diagram show the location of 3' ends by vertical arrows. For
each location, the composition of non-encoded oligonucleotide tails is shown. The numbers in
parentheses indicate the number of identical clones. Non-encoded nucleotides are coloured in
blue (A), red (T) or green (G or C).
Fig. S12: Accumulation of low molecular weight fragments and 5.8S precursors in trl
mutants.
Total RNA isolated from seedlings of WT, trl-1, trl-2 and mtr4 mutants was separated on
denaturing 6% polyacrylamide gels and hybridized with a random-primed DNA probe
complementary to P-P' (S1RP) (a) and the oligoprobe S5 (b) to visualize low molecular weight
fragments derived from the 5' ETS and 5.8S precursors, respectively. The detected precursors
and degradation intermediates are labelled on the right. Triangles mark degradation
intermediates specifically observed upon downregulation of TRL. Hybridization with an
oligoprobe complementary to the 7SL RNA is shown as loading control.
Table S1: List of primers used in this study. All primers are from 5' to 3'.