(Received for publication, August 30, 1995; and in revised form, September 18, 1995)
From the
In eukaryotes, nascent rDNA and 5 S rRNA gene transcripts undergo 3`-end processing after termination. Mutations in which terminator sequences in these ribosomal RNA genes are deleted completely result in highly unstable transcripts, which are not properly processed and integrated into stable ribosome structure. Mutations that retard RNA processing by extending the 3` external transcribed spacer or by introducing additional secondary structure in the spacers have a similar effect on stable transcript integration. The results indicate that proper termination coupled with efficient rRNA processing acts as a ``quality control'' process, which helps to ensure that only normal rRNA precursors are effectively processed and assembled into active ribosomes.
It is generally assumed that the termination of RNA
transcription, even when genes are tandemly arranged, is largely an
economic consideration, which leads to a conservation of cellular
energy and allows for individual gene regulation. The wide distribution
of often very large introns, which are rapidly discarded after
transcription and RNA splicing, appears to make this measure less
important, and it is surprising that with tandemly arranged highly
repeated genes such as the rDNA, in which transcribed spacers actually
can be longer than the non-transcribed regions, the termination signal
is repeated (1, 2) and even fail safe copies are
present(3, 4) . Furthermore, in another tandemly
arranged family, the genes encoding the 5 S rRNA, termination occurs
only a few nucleotides after the 5 S rRNA sequence with a complex
processing scheme essential for a mature RNA
product(5, 6, 7) . Both features clearly are
not consistent with an economical use of cellular energy but still are
widely conserved in eukaryotic organisms. In recent studies on
eukaryotic ribosome biosynthesis and rRNA processing, we have been
expressing mutant genes in vivo in order to identify important
structural features in the transcripts that contribute to rRNA function
and ribosome assembly. Efficiently expressed ``tagged'' RNA
systems have been developed for both the yeast 5 S rRNA gene (8, 9) and rDNA expression(10, 11) .
These can result in cellular RNA and ribosome populations that are
50-90% mutant with no adverse effects on cellular growth or
function. In the course of these studies, we have been making changes
in the 3` external transcribed spacers (3`-ETS) ( As previously reported (8, 11, 12) and illustrated by the example
analyses in Fig. 1, when a yeast rDNA transcriptional unit or a
gene encoding the 5 S rRNA is inserted in a high copy shuttle vector
and expressed in yeast after cell transformation, an efficient and even
preferential expression of plasmid-encoded RNA is observed with
50-90% of the RNA population being derived from plasmid-encoded
transcripts. With direct 5 S rRNA quantification (Fig. 1A), the electrophoretic marker shows that
80-90% of the cellular 5 S RNA is mutant (lane b).
Similarly, when 3`-end termini are mapped using S
Figure 1:
Effect of transcript termination on
rRNA stability in ribosome biogenesis. A, a truncated 5 S rRNA
gene without its 3`-end termination region was synthesized by PCR
amplification (9) using a primer complementary to the 3`-end of
the mature 5 S rRNA sequence and a plasmid DNA template (pYF5A99)
containing a structurally marked (A99) 5 S rRNA gene
sequence(8) . The mutant gene was cloned into pYF404, a high
copy (30-40 copies/cell) yeast shuttle vector(22) , the
sequence was confirmed by DNA sequencing, and the recombinant
(pYF5A99
As indicated in Fig. 2, the normal 5 S rRNA transcript (a, b) is extended by about 12 nucleotides,
terminating in a polyuridylic acid cluster of 4-5
residues(5, 6, 7) , this extension being
rapidly removed during maturation. As illustrated with two examples in Fig. 3, this spacer sequence could be modestly altered, both
with respect to the nucleotide sequence and length without an effect on
the mature rRNA product. For example, when the spacer was abbreviated
to only a poly(U) cluster (lane c), which constitutes the
basic termination signal(5, 6, 7) , or
modestly lengthened to 24 nucleotides with poly(C) clusters (lane
d), the plasmid-derived molecules continued to constitute
80-90% of the cellular 5 S rRNA population. In sharp contrast,
however, when the poly(U) cluster was substantially displaced (lane
e) with a long spacer (490 base pairs), a mature product was again
absent as was observed earlier (Fig. 1) with a deleted
termination signal.
Figure 2:
Summary of 3` external transcribed
sequences in mutant 5 S rRNA gene transcripts. The solid line corresponds to the mature 5 S rRNA, and the termini are identified
to correspond with the sample lanes in Fig. 3.
Figure 3:
Effect of 3` external transcribed
sequences on 5 S rRNA stability. Altered 3`-ETS regions in the S.
cerevisiae 5 S rRNA gene (Fig. 2) were synthesized by PCR
amplification using primers containing the mutant sequence but still
complementary to the 3`-end of the normal 5 S rRNA sequence and a
plasmid DNA template (pYF5A99) containing a structurally marked (A99) 5 S rRNA gene sequence(8, 9) . The
mutant genes were again cloned in pYF404, the sequences were confirmed
by DNA sequencing, and the recombinants were used to transform
LEU2-deficient S. cerevisiae cells. Whole cell RNA was
prepared from exponentially growing transformants (lanes
c-g) and fractionated by gel electrophoresis (8) as
described in Fig. 1. RNA from untransformed cells and cells
transformed with pYF5A99 were included in lanes a and b, respectively; the positions of the normal and
plasmid-derived 5 S rRNAs are indicated by arrows.
Because the spacer sequence appeared not to be
directly critical to RNA product stability and ribosomal integration,
other features were examined, namely secondary structure and length. As
also illustrated in Fig. 2and Fig. 3, both of these
proved to be important factors in RNA stability. When the sequence was
altered to insert a small (5 base pairs) hairpin structure in the
spacer (lane f) only about 50% of the mature RNA was of
plasmid origin, and when the spacer was more substantially lengthened
to 41 nucleotides (lane g), only about 10% of the mature RNA
was of plasmid origin. Taken together, the results shown in Fig. 1and Fig. 3indicate that termination followed by
efficient processing is essential for transcript stability. Presumably,
with inefficient processing or no processing at all, the nascent
transcript is not integrated into ribosomal structure and is
susceptible to cellular ``housekeeping'' degradation, which
may even be mediated, at least in part, by the processing enzymes. In a
number of organisms(13, 14, 15) , 5 S rRNA
processing has been shown to be dependent, at least in part, on
exonuclease cleavage, and while the details are less clear, nucleolar
RNA processing is also dependent, at least in part, on exonuclease
trimming (see (16) ). It appears, therefore, that when the
termination site is too distant from the mature RNA or encumbered with
secondary structure, processing is sufficiently retarded or eliminated,
resulting in transcript degradation without maturation. Indeed, the
significance of termination as a factor in quality control is not
likely restricted to pol I or III transcripts. Although the termination
signal has not been clearly defined with pol II transcripts (17) , mRNA instability often has been linked to changes in the
termination region. When many mRNAs are not polyadenylated (see (18) and (19) for reviews), rapid turnover is
frequently reported, and there is evidence that the function of
intrinsic terminators is coupled to the functioning of the 3`-end
maturation signals(20) , indicating that defects which prevent
cleavage of the nascent transcript also prevent the functioning of the
downstream terminators. A conclusion cannot be drawn until pol II
termination is further clarified. In the interim, the observations made
here clearly show that, at least for pol I and III transcripts, proper
termination appears to help in ensuring that only normal precursors are
being assembled into ribosomes. Furthermore, when the present results
are taken together with recent reports of relationships between
enhancer and terminator sequences in rDNAs (see (21) ), they
may even indicate that correct termination plays a role in coordinate
regulation through the same degradation mechanism.
)and
termination regions to determine specific features that affect 3`-ETS
processing (e.g.(12) ). The changes have included
controls in which the termination signals have been altered or removed
entirely. Here we report that the yields of the mature RNAs are
severely reduced when the termination signals are compromised, an
observation which indicates that proper termination coupled with RNA
processing can be an important component of a cell's control on
the quality of its RNA transcripts.
nuclease
digestion (Fig. 1B) and a mutant RNA-specific
probe(12) , the termination sites, processing intermediates,
and mature 25 S rRNA are all clearly evident (lane d). In
striking contrast, however, when the termination signals are deleted
using PCR-mediated targeted mutagenesis(9, 12) ,
almost no plasmid-derived RNA is evident. As shown in Fig. 1,
steady state analyses of the cellular 5 S RNA population (Fig. 1A) indicate that, without normal termination (lane c), the plasmid-derived transcripts are very unstable
with no plasmid-derived RNA being observed after methylene blue
staining. Similarly (Fig. 1B), only trace amounts or no
plasmid-derived 25 S rRNA or processing intermediates are evident (lane c) when unterminated rDNA transcripts are characterized
by S
nuclease digestion studies. Both results clearly
demonstrate a striking instability in nascent transcripts that are not
properly terminated, with no mature plasmid-derived RNA being detected.
T) was used to transform LEU2-deficient Saccharomyces
cerevisiae strain (AH22) as described by Hinnen et
al.(23) . Whole cell RNA was prepared (8, 9) from exponentially growing pYF5A99
T
transformed cells (lane c) as well as untransformed (lane
a) and pYF5A99-transformed cells (lane b), fractionated
on 12% non-denaturing polyacrylamide gels, and stained with methylene
blue as described previously(8, 9) . The positions of
the normal and plasmid-derived RNAs are indicated by arrows. B, a truncated rDNA 3`-ETS region lacking the three
``Salbox''-like termination signal repeats (12) was
synthesized by PCR amplification (24) using a primer
complementary to the sequence immediately upstream of the terminator
repeats and a plasmid template (pFL20/Sp25NotI) containing Schizosaccharomyces pombe rDNA with a unique NotI
cloning site in the 25 S rRNA sequence(10, 12) . The
mutated 3` was substituted in pFL20/Sp25NotI using the unique NotI and PvuII sites(12) , resulting in a
rDNA construct (pFL20Sp
3T), which contained no known termination
signal, and the sequence was confirmed by DNA sequencing. Whole cell
RNA was prepared from exponentially growing pFL20Sp
3T transformed S. pombe cells, strain h
leu1-32 ura
4-D18 (lane c), as well as untransformed cells (lane
b) and cells transformed with normal plasmid, pFL20/Sp25 NotI (lane d) and from E. coli cells (lane e). RNA aliquots (20 µg) were incubated with labeled
plasmid-specific probe, which was complementary to the pFL20/Sp25 NotI sequence (12) at 30 °C for 12 h, and the
hybrids were digested with 100 units of S
nuclease at 37
°C for 30 min. After extraction with phenol/chloroform, the
fragments were fractionated on a 6% polyacrylamide sequencing gel
together with standard dideoxy sequencing reaction products (A, G, T,
C) as residue markers. The positions of the normal 25 S rRNA 3`-end,
major processing intermediate (+21), and major
termination (T1) sites are indicated at the right.