Cell, Vol. 117, 311–321, April 30, 2004, Copyright 2004 by Cell Press
Identification of a Human Endonuclease Complex
Reveals a Link between tRNA Splicing and
Pre-mRNA 3? End Formation
splice junctions, but the 3? splice site is invariably lo-
cated in a bulged loop (Baldi et al., 1992).
The removal of introns from pre-tRNA is an enzymatic
reaction that requires the activity of several different
proteins (reviewed in Abelson et al. ). These en-
zymes have been most intensively investigated in Ar-
chaea and yeast. The first step is carried out by an
evolutionarily conserved tRNA splicing endonuclease
that recognizes and cleaves precursor tRNA at the 5?
and 3? splice sites (Trotta et al., 1997). In yeast, the 5?
and 3? exons are ligated by a tRNA ligase through a
series of enzymatic reactions that lead to joining of the
two exons with a 2? phosphate at the splice junction
(Westaway et al., 1988; Phizicky et al., 1986). This un-
usualtRNAintermediate isthenprocessedby a2?phos-
photransferase, yielding a mature tRNA (Culver et al.,
Yeast tRNA splicing endonuclease is a heteromeric
complex of four subunits encoded by the SEN2, SEN34,
SEN54, and SEN15 genes (Rauhut et al., 1990; Trotta et
al., 1997). All four subunits are present at low levels and
are essential for cell viability (Trotta et al., 1997). The
structure and function of the factors of the yeast tRNA
endonuclease complex have been suggested from a
number of experimental results. First, strong sequence
conservation of the yeast Sen2p and Sen34p to the
homotetrameric archaeal enzyme suggested that these
two subunits each contained a distinct active site for
cleavageat the5?and 3?sites.Consistentwith thisview,
a mutation in Sen2p resulted in a defect in cleavage of
the 5? splice site (Ho et al., 1990), whereas a mutation
in a conserved histidine residue in Sen34p resulted
in a defect in cleavage of the 3? splice site (Trotta et
al., 1997). Second, two-hybrid analysis demonstrated
strong interaction between Sen2p and Sen54p and be-
tween Sen34p and Sen15p (Trotta et al., 1997). Struc-
tural studies with the homotetrameric archaeal tRNA
endonuclease suggested that the strong interaction be-
tween Sen2p-Sen54p and Sen34p-Sen15p is mediated
by a conserved carboxyl-terminal ? sheet interaction
(Lykke-Andersen and Garrett, 1997; Li et al., 1998). Fi-
nally, sequence alignment of heterologous subunits
Sen54p and Sen15p to the archaeal endonuclease re-
vealeda conservedstructural elementnear thecarboxyl
terminus required for dimerization of the two yeast het-
erodimers Sen54p-Sen2p and Sen15p-Sen34p (Lykke-
Andersen and Garrett, 1997; Li et al., 1998). Together,
these results led to a model for the configuration of
the yeast tRNA splicing endonuclease (Li et al., 1998;
Abelson et al., 1998).
Preliminary studies suggest a common mechanism
for tRNA splicing throughout evolution. For example,
extracts derived from human cell lines were reported to
carry out accurate tRNA splicing under conditions in
which the yeast tRNA splicing endonuclease is active
(Laski et al., 1983; Standring et al., 1981). Furthermore,
partially purified tRNA splicing endonuclease from Xen-
opus laevis germinal vesicles was shown to recognize
and accurately cleave yeast pre-tRNA, forming two half
molecules and an intron (Gandini-Attardi et al., 1990;
Sergey V. Paushkin, Meenal Patel, Bansri S. Furia,
Stuart W. Peltz, and Christopher R. Trotta*
100 Corporate Court
South Plainfield, New Jersey 07080
tRNA splicing is a fundamental process required for
cell growth and division. The first step in tRNA splicing
is the removal of introns catalyzed in yeast by the
tRNA splicing endonuclease. The enzyme responsible
for intron removal in mammalian cells is unknown.
We present the identification and characterization of
the human tRNA splicing endonuclease. This enzyme
consists of HsSen2, HsSen34, HsSen15, and HsSen54,
homologs of the yeast tRNA endonuclease subunits.
Additionally, we identified an alternatively spliced
isoform of SEN2 that is part of a complex with unique
RNA endonuclease activity. Surprisingly, both human
endonuclease complexes are associated with pre-
mediated depletion of SEN2 exhibited defects in
maturation of both pre-tRNA and pre-mRNA. These
and pre-mRNA 3? end formation, suggesting that the
endonuclease subunits function in multiple RNA-
Maturation of cellular RNAs is critical for regulation of
are generated from large precursors via a series of pro-
cessing steps. For example, nascent pre-mRNAs un-
dergo splicing, capping, and generation of 3? ends
by endonucleolytic cleavage and polyadenylation. The
maturation of precursor transfer RNA (pre-tRNA) re-
quires several steps that include 1) removal of both the
5? leader by RNase P (Xiao et al., 2002; Frank and Pace,
1998) and the 3? trailer by ELAC2 (Takaku et al., 2003),
2) addition of the CCA trinucleotide to the 3? end, and 3)
and Phizicky, 2003). In addition, several tRNAs contain
introns that must be removed to produce a mature
Intron-containing pre-tRNAs are found in a variety of
otes, approximately 25% of all tRNA genes contain in-
trons (Trotta et al., 1997), whereas, in humans, only 6%
of tRNA genes contain introns (Lowe and Eddy, 1997).
All eukaryotic tRNA introns are 14–60 nucleotides in
length and interrupt the anticodon loop one nucleotide
3? to the anticodon (Ogden et al., 1984). Among all yeast
pre-tRNAs, there is no sequence conservation at the
pus and yeast enzymes appear to fix the sites of cleav-
age by recognition of local structures at the intron-exon
boundaries (Baldi et al., 1992; Fabbri et al., 1998).
Although there is evidence that the mechanism of
and higher eukaryotes, the enzymes responsible for the
maturation of pre-tRNA in humans are unknown. Here,
we present the isolation and characterization of human
fied a distinct endonuclease complex resulting from al-
ternative splicing of the SEN2 subunit. This complex
differs from tRNA endonuclease complex by protein
composition and the ability to process pre-tRNA. Fur-
thermore, the endonuclease complex associates with
factors required for cleavage/polyadenylation ofmRNAs,
suggesting a previously undiscovered biochemical link
between pre-tRNA splicing and formation of the 3? end
of messenger RNAs.
this hypothesis, we performed PCR analysis of cDNA
libraries obtained from different human tissues using
oligonucleotides flanking exon 8 and monitored the
presence of either full-length SEN2 or SEN2 lacking
exon 8 (HsSen2?Ex8). All tissues examined harbored
both isoforms of SEN2 (data not shown). Using a human
multiple tissue expression array, we profiled the ex-
pression of HsSen2 and HsSen2?Ex8 RNAs in human
tissues and cancer cell lines. Northern blot analysis
was performed with oligonucleotides specific for either
SEN2 or SEN2?Ex8. The results demonstrated that both
mRNAs are ubiquitously expressed at very low levels in
all tissue types (data not shown).
The Human Endonuclease Forms Two
Functionally Distinct Isoforms
To determine whether the human homologs of the yeast
endonuclease subunits function as part of a tRNA splic-
ing complex, a method was developed for the purifica-
lines expressing His-Flag-tagged human homologs of
the active site subunits HsSen2 or HsSen34 as well as
the alternatively spliced subunit HsSen?Ex8. Proteins
from total cell extracts of the stable cell lines were puri-
fied by affinity chromatography using anti-FLAG M2 af-
finity resin followed by Ni-NTA agarose resin. Bound
to cleave yeast pre-tRNAPhe. The results demonstrated
that protein complexes isolated from cells expressing
either His-Flag-HsSen2 or His-Flag-HsSen34 accurately
cleaved pre-tRNAPheto yield 5? exon, 3? exon, and intron
(Figure 2B, lanes 4 and 5). The efficiency of cleavage
was similar to that of yeast tRNA splicing endonuclease
(Figure 2B, compare lanes 4 and 5 with lane 2). Purifica-
tion of cleavage activity was dependent upon expres-
sion of an epitope-tagged subunit, as proteins purified
from untransfected 293 cells did not cleave pre-tRNA
(Figure 2B, lane 1). Taken together, these results clearly
demonstrate that HsSen2 and HsSen34 are orthologs
of the yeast tRNA splicing endonuclease subunits and
that the enzyme for cleavage of pre-tRNA is evolution-
The endonuclease complex harboring the His-Flag-
as described above. Surprisingly, the His-Flag-HsSen2-
?Ex8-containing complex retained the ability to cleave
precursor tRNA, but the fidelity and accuracy of cleav-
age was severely compromised, resulting in cleavage
at only the 3? splice site. Moreover, the HsSen2?Ex8-
containing complex was unable to release the intron
from the pre-tRNA (Figure 2B, lane 3). In addition, there
was a minor cleavage event within the intron of tRNAPhe,
resulting in two products migrating at approximately 53
and 42 nucleotide positions (Figure 2B, lane 3, asterisk).
This minor cleavage product is not detected with other
precursor tRNAs (data not shown). Thus, pre-tRNA is
the endogenous substrate for the HsSen2-containing
complex but not for the HsSen2?Ex8-containing com-
plex. This important observation suggests that the gene
for the human endonuclease subunit SEN2 can encode
two distinct active site-containing proteins, each with
different RNA cleavage specificities.
Human Homologs of the Yeast tRNA Splicing
To identify human homologs of the tRNA splicing endo-
nuclease subunits, we performed a BLAST search of
the human protein database using protein sequences
of all four subunits of the S. cerevisiae tRNA splicing
endonuclease. We found potential human homologs for
three subunits, SEN54, SEN2, and SEN34 (Figures 1A–
1C) but were unable to identify a human homolog of
yeast SEN15. Human Sen54 (HsSen54) has a predicted
molecular mass of 58 kDa, and amino acid conservation
between the yeast and human Sen54p was restricted to
the amino- and carboxyl-terminal regions of the protein
(Figure 1A). Human Sen2 (HsSen2) is predicted to be 51
kDa, larger than its yeast counterpart, and shows a high
degree of similarity only in the active site domain (Figure
1B). Conversely, the yeast and human Sen34 (Figure 1C)
are highly homologous throughout the entire protein.
Importantly, sequence alignments between yeast and
human Sen2 and Sen34, the two subunits harboring the
endonuclease active sites (Trotta et al., 1997), demon-
sponding to the active sites of Sen2 and Sen34. These
findings indicate a remarkable conservation between
The Human Sen2 Transcript Is Alternatively
Spliced to Form at Least Two Distinct
We next determined whether the putative human SEN2
and SEN34 genes encode subunits of the tRNA splicing
endonuclease complex. To accomplish this, we isolated
the human SEN2 and SEN34 cDNAs. Surprisingly, se-
quencing of SEN2 clones produced by PCR amplifica-
tion from human cDNA libraries identified a variant that
lacked 57 nucleotides. This deletion corresponds pre-
cisely to exon 8 of the SEN2 genomic DNA sequence
(Figure 2A), suggesting that this was an alternatively
spliced form of SEN2.
The result described above raised the possibility that
Human tRNA Splicing Endonuclease
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