Download 1-Tubulin mRNAs Are Specified by the First 13

MOLECULAR AND CELLULAR BIOLOGY, Mar. 1988, p. 1224-1235
Vol. 8, No. 3
0270-7306/88/031224-12$02.00/0
Copyright © 1988, American Society for Microbiology
Autoregulated Changes in Stability of Polyribosome-Bound
1-Tubulin mRNAs Are Specified by the First 13
Translated Nucleotides
TIM J. YEN, DAVID A. GAY, JOEL S. PACHTER, AND DON W. CLEVELAND*
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe Street,
Baltimore, Maryland 21205
Received 16 October 1987/Accepted 15 December 1987
The expression of tubulin polypeptides in animal cells is controlled by an autoregulatory mechanism whereby
increases in the tubulin subunit concentration result in rapid and specific degradation of tubulin mRNAs. We
have now determined that the sequences that are necessary and sufficient to specify mouse I-tubulin mRNAs
as substrates for this autoregulated instability reside within the first 13 translated nucleotides (which encode the
first four 13-tubulin amino acids Met-Arg-Glu-Ile). This domain has been functionally conserved throughout
evolution, inasmuch as sequences isolated from the analogous region of human, chicken, and yeast j-tubulin
mRNAs also confer autoregulation. Further, for an RNA to be a substrate for regulation, not only must it carry
the 13-nucleotide coding sequence, but it must also be ribosome bound and its translation must proceed 3' to
codon 41.
Microtubules are protein polymers which participate in a
large spectrum of cellular functions, including formation of
mitotic and meiotic spindles, transport of cellular components, establishment of cell shape during programmed differentiation such as neurite outgrowth and flagellar assembly, and, in concert with intermediate filaments and actin
filaments, construction of the animal cell cytoskeleton.
These structures are composed principally of heterodimers
of a- and P-tubulin polypeptides for which (unlike most
nonassembling cellular components) a rapid, dynamic equilibrium exists between the subunit and the polymeric form
(21).
A priori, the participation of microtubules in so many
important cellular events suggests the need for a sensitive
mechanism(s) for regulating the synthesis of both a and I8
tubulins. In fact, for animal cells we now know that the
control of tubulin expression is exercised at two general
levels. The first level is selective transcriptional activation
during cell differentiation of one or more of the -6 to 7
functional genes that encode either subunit (7). The second
level is establishment of the proper quantitative level of
expression of the selected gene or genes. For this second
regulatory aspect, beginning with the initial work of Ben
Ze'ev et al. (1) and followed by subsequent efforts from this
laboratory (8, 10, 11, 16, 24, 33, 35) and by Kirschner's
group (5, 6), it has become clear that most animal cells
rapidly and specifically depress the synthesis of both a- and
P-tubulin polypeptides in response to increases in the intracellular pool of tubulin subunits. This decrease in synthesis
(induced either by treatment with microtubule-destabilizing
drugs such as colchicine and nocodazole or by direct microinjection of tubulin subunits) is the result of a posttranscriptional mechanism that alters the stability of cytoplasmic
tubulin mRNAs after changes in the concentration of unpolymerized tubulin subunits. Most recently, we have used
DNA transfection and inhibitors of protein synthesis (33) to
demonstrate that only tubulin mRNAs which are attached to
polyribosomes are autoregulated.
*
One obvious general question that remains to be answered
is how tubulin RNAs are specifically targeted to be substrates for autoregulated changes in mRNA stability. By
transfecting plasmid constructs which carry different regions
of the chicken ,2-tubulin gene, we have previously localized
a regulatory element to the 5' 106 nucleotides of exon 1 in
chicken ,2 mRNA (16). In the present study, we have
extended these findings to demonstrate unambiguously that
sequences necessary and sufficient to confer autoregulation
are contained within a 13-nucleotide segment that encodes
the first four amino acids of 1B tubulin. Further, we show that
for an RNA to be a substrate for regulation not only must it
carry the 13-nucleotide sequence, but it must also be ribosome bound, and translation of the RNA must also proceed
3' to codon 41.
MATERIALS AND METHODS
Construction of hybrid genes containing amino-terminal
1-tubulin-coding sequences linked to tk. (i) MT-C16-tk. To
construct the MT-C16-tk gene, we used a two-step process.
A 1.8-kilobase metallothionein (MT) promoter fragment was
recovered from clone pMTP,2A5' (16) by sequential digestion
with EcoRI, mung bean nuclease (to create a blunt end), and
HindIII. Herpes simplex virus (HSV) thymidine kinase
(tk)-coding sequences were excised from plasmid pMK (3)
by digestion with BgIII (which cleaves in the 5' untranslated
region of tk) and EcoRI (which cleaves within the 3'-flanking
sequences), gel isolation, and subcloning between the EcoRI
and BamHI sites of pUC18. The tk segment was then
excised with EcoRI and Hindlll, gel isolated, and cloned
with the MT promoter fragment in a trimolecular ligation
into pBR322 which had been digested with EcoRI and NdeI
(blunted with mung bean nuclease). To obtain a restriction
fragment carrying codons 1 to 16 of 132 tubulin, we again
started from clone pMT132A5', a plasmid carrying a copy of
the chicken 12 tubulin gene (29, 41) from which all tubulin
sequences 5' to the ATG translation initiation were deleted
by BAL 31 exonuclease digestion (16). By digesting the
clone with HaeII (which cleaves within codon 17), treating it
with mung bean nuclease to create a blunt end, and finally
Corresponding author.
1224
VOL. 8, 1988
NUCLEOTIDES SPECIFYING 1-TUBULIN AUTOREGULATION
digesting it with HindIlI (which cleaves 11 bases 5' to the
P-tubulin ATG), we obtained a fragment carrying only the
first 49 translated nucleotides of ,2 tubulin. To create MTC16-tk, this fragment was subcloned between the HindlIl
and NruI sites of the MT promoter and HSV tk vector
(which were cleaved within the MT 5' untranslated region
[68 bases 3' to the major transcription initiation site] and at
codon 97 of tk, respectively). The RNA transcript of MTC16-tk should be translated into a fusion protein consisting
of 16 amino-terminal ,-tubulin residues and 278 amino acids
of tk. The sequence of the P2/tk junction was verified by
subcloning the relevant segment from the final plasmid into
M13 followed by dideoxy sequencing (36).
(ii) MT-M16-tk, MT-H18-tk, and MT-Y16-tk. Fusion genes
whose RNAs encoded a fusion polypeptide consisting of 16
to 18 amino-terminal codons of fi tubulin linked to 278 amino
acids of tk were constructed as follows. Mouse mP5 (26) and
human H1l (encoded by gene M40; 25) tubulin sequences
were obtained from mouse or human cDNA clones, subcloned into pUC9 after being isolated from Xgtll libraries
(40; K. F. Sullivan and D. W. Cleveland, unpublished), and
the Saccharomyces cerevisiae TUB2 sequence was derived
from plasmid pJT179, which carries the unique (intronless)
S. cerevisiae ,-tubulin gene (32). To remove the 5' untranslated sequences from each of these tubulin genes, we digested each plasmid with EcoRI, which cleaved each at
-150 to 300 bases 5' to the translation initiation codon.
Double-stranded deletions were produced by successive
treatment with exonuclease III followed by mung bean
nuclease digestion. The DNAs were subsequently digested
with BglII, which cleaved each at a site within the tubulin coding region (between 200 and 250 nucleotides downstream of the initiating ATG), and subcloned into BamHIHincII-digested M13mpl8. The deletions were screened by
DNA sequencing. Clones M13MPTUB, M13HITUB, and
M13Y,BTUB (from mouse, human, and S. cerevisiae DNA,
respectively) contained deletions that extended 2, 3, and 11
nucleotides, respectively, 5' to the initiating ATG.
These intermediates were then used to initiate a second
phase of deletions, this time involving the processive removal of tubulin-coding sequences from the 3' end. After
cleavage at the unique EcoRI site located in the M13
polylinker 3' to the tubulin sequences, exonuclease III and
mung bean nuclease digestion was again used to generate a
set of fragments carrying random 3' deletions. The DNAs
were digested at the unique Bglll site within the M13
sequences, and the collection of fragments was then ligated
to a BgIII-HincII fragment of fusion vector M13-NruItk.
This vector was itself constructed by ligating the 1.7-kilobase NruI-EcoRI fragment of tk (that contained the coding
sequence for tk beginning at codon 97 and extending through
the 3'-flanking region [Fig. 1A]) into M13mpl8 that had been
digested with EcoRI and XbaI (the latter had been blunted
with mung bean nuclease). M13-Nrultk was then digested
with BglII (within the M13 coding sequence) and with Hincll
(in the polylinker), and the 9-kilobase fragment (containing
the portion of M13 complementary to that carried by the
tubulin 3' deletions) was isolated and ligated to the tubulin
deletions. (The strategy of using the M13 BglII site allowed
the direct selection of recombinants, since the BglII-Hinclldigested fusion vector lacked an essential portion of M13
sequences which could only be provided by the fragments
carrying tubulin sequences with random 3' deletions.)
Plaques were randomly selected and screened by dideoxy
DNA sequencing to determine the extent of 3' deletions and
the precise sequence at the junction between tubulin and tk.
1225
Although the tk-coding sequences began at the desired NruI
site, the actual fusion with tubulin was separated by six
bases of M13 linker DNA. (Only tubulin deletions ending
after the first nucleotide of a tubulin codon created in-frame
fusions with the truncated tk gene.)
To form the final plasmids, replicative-form DNA from
appropriate M13 deletions was cleaved with EcoRI at the
EcoRI site in the 3'-flanking region of tk. The DNA was
blunted with Klenow, cleaved with HindIII (at the M13
linker site 5' to the tubulin-tk fusion) and ligated into plasmid
DNA from pMTtk that had been digested with NdeI (at the
site within the pBR322 vector), blunted with Klenow, and
finally digested with HindIII (within the MT 5' untranslated
region).
Construction of cI2A2 and cI2A3/4. Site-directed mutagenesis was used to construct cP2A2 and cp2A3/4. A 700base-pair SmaI fragment which contained exon 1 of the
authentic c,B2 gene was subcloned into M13mpl8, and recombinants were selected which had the insert oriented such
that the coding strand of exon 1 was also the plus strand of
the phage. OligoA2 (5'GTGCACGATCTCCATGATGCC
GGT3') and oligoA3/4 (5'CTGGATGTGCACACGCATGA
TGCC3') were synthesized by using a DNA synthesizer
(Applied Biosystems model 380A) and purified by high-pressure liquid chromatography. Mutagenesis was performed as
described previously (44) and as modified elsewhere (23).
After the desired deletion was identified by DNA sequencing, the SmaI fragment from the M13 recombinant was
subcloned in the correct orientation into plasmid pcP2ASma
at its unique SmaI site to create pc,32A2Sma and pcp2A3/
4Sma. pc32ASma was generated by cutting pcP2 with SmaI
and religating the vector portion. Since there were three
SmaI sites within c132, reintroduction of the mutagenized
SmaI 700-base-pair fragment into pc32ASma did not fully
regenerate an intact gene. To restore the missing 5'-flanking
sequences in these last two constructs, a 1-kilobase AatII
fragment which spanned the two 5'-proximal SmaI sites was
isolated from authentic c,B2 and introduced into pcp32A2Sma
and pcp2A3/4Sma which had been digested with AatII. The
final constructs pcP2A2 and pcf32A3/4 were identical to the
authentic c,32 gene, except for the presence of the corresponding deletions within exon 1.
Si probes. To detect RNAs transcribed from either transfected or endogenous genes, the following DNA probes were
used in S1 nuclease protection analyses. All probes were 5'
end labeled with polynucleotide kinase after being digested
with the appropriate restriction endonuclease and phosphatase treatment.
(i) Ribosomal RNA. To analyze the level of endogenous
18S rRNA, pl8S (18), a plasmid carrying a portion of the
mouse 18S rDNA gene, was linearized at the BamHI site
that lies 600 bases 3' to the mature 5' border of 18S rRNA.
Hence, a 600-base protected fragment was expected after
hybridization to 18S rRNA and S1 nuclease digestion.
(ii) Mouse j5-tubulin mRNA. To detect RNAs from the
endogenous mouse m15 gene, a probe was prepared from a
pUC9 subclone of m,B5 sequences isolated from a Xgtll
cDNA library (40). This plasmid was linearized at the unique
XhoI site that lies 355 nucleotides from the 5' border of the
cloned cDNA. S1 nuclease analysis of m15 RNAs with this
probe should yield a protected fragment of 355 bases.
(iii) Chicken (2-tubulin mRNAs (c32, c02A2, and
cp2A3/4). To detect RNAs transcribed from transfected
chicken ct2, we used a probe from a cDNA clone (pT2; 9) of
c,B2. Since this cDNA clone actually derives from an RNA
that was initiated from a very minor cP2 transcription
1226
YEN ET AL.
initiation site that lies 29 bases 5' to the major site of
transcription initiation (41), it contains -24 bases of sequence 5' to the major site of initiation. After digestion and
end labeling at the unique BglII site at codon 84, an Si
resistant fragment of 315 nucleotides was expected for
spliced cP2 RNAs that were initiated at the major transcription initiation site. For RNA with deletions at codons 2 or 3
and 4, protected fragments of 246 and 240 nucleotides,
respectively, were expected after probe cleavage at the site
of the deletion.
(iv) MT-C-tk, MT-H-tk, MT-M-tk, and MT-Y-tk series. To
detect RNAs from the chicken (MT-C-tk), human (MT-Htk), mouse (MT-M-tk), or yeast (MT-Y-tk) series of deletion
constructs fused to tk, plasmid DNA from each gene was 5'
end labeled at the EcoRV site in the HSV tk-coding region
(49 nucleotides from the tubulin-HSV tk fusion junction).
The size of the predicted fragments included 49 nucleotides
of tk and linker sequences, the number of P-tubulin nucleotides, and 68 bases of MT 5' untranslated region.
(v) MTtk mRNA. The MTtk gene was 5' end labeled at the
NruI site (codon 97). MTtk mRNAs initiating from the MT
promoter were expected to yield an S1 resistant product of
449 nucleotides. This product included 348 nucleotides of
HSV tk sequence (292 nucleotides of coding sequence and 56
nucleotides of 5' untranslated leader), 33 nucleotides of M13
polylinker, and 68 nucleotides of MT 5' untranslated sequence.
DNA transfections. Mouse Ltk- cells were transiently
transfected by using a modified DEAE-dextran procedure as
described by Lopata et al. (28). In all cases, parallel dishes of
cells were transfected with 8.0 ,ug of the complete c,B2 gene
or with each engineered construct. At 30 to 38 h posttransfection, duplicate dishes of transfected cells were incubated
for the final 6 h of culture in normal medium (DMEM; 4,500
mg of glucose per liter, 10% fetal calf serum, 290 mg of
glutamine per liter) or in the same medium containing 10 ,uM
colchicine.
RNA isolation. Cytoplasmic RNA was isolated by a modification by Favaloro et al. (15). Cells were washed once with
5 ml of ice-cold phosphate-buffered saline, and 300 ,ul of lysis
buffer (0.14 M NaCl, 1.5 mM MgCl2, 10 mM Tris [pH 8.6],
0.5% Nonidet P-40, 10 mM vanadyl-ribonucleoside complexes) was added to the monolayer of cells. Cells were
scraped with a rubber policeman and transferred to 1.5-ml
Microfuge tubes prechilled to 4°C. Lysis was achieved by
gentle vortexing for 10 s, and the cell extract was centrifuged
for 4 min in a Microfuge at 4°C to remove nuclei. The
supernatant was removed and diluted with an equal volume
of 2x proteinase K buffer (0.2 M Tris hydrochloride [pH
7.5], 25 mM EDTA, 0.3 M NaCl, 2% sodium dodecyl
sulfate). Proteinase K was added to 400 ,ug/ml, and the cell
lysate was incubated at 37°C for 30 min. After this time, the
lysate was phenol-chloroform extracted, chloroform extracted, and then precipitated with 1/10 volume of 3M
sodium acetate (pH 5.2), and 2.5 volumes of ethanol. The
resulting RNA pellet was suspended in diethylpyrocarbonate-treated water, quantitated by A260, and stored frozen at
-800C.
Si nuclease analysis. An excess of double-stranded DNA
probe (0.02 pmol) was mixed with 2.5 ,ug of cytoplasmic
RNA (to measure 18S rRNA, only 0.25 ng was used),
lyophilized to dryness, and suspended in 20 ,lI of a solution
containing 80% formamide, 0.4 M NaCl, 40 mM PIPES
[piperazine-N,N'-bis(2-ethanesulfonic acid)] (pH 6.4), and 1
mM EDTA. After being heated to 90°C for 5 min, the
hybridization reactions were incubated for 12 to 16 h at
MOL. CELL. BIOL.
either 61°C (for pl8S-5', pmP5, and chicken P2 gene) or 59°C
(for cp2A2m, cp2A3/4, MTtk, and MT-Y-tk, MT-C-tk, MTH-tk, and MT-M-tk plasmids), diluted 10-fold by addition to
ice-cold S1 nuclease buffer (0.2 M NaCl, 30 mM sodium
acetate [pH 4.5], 5 mM ZnC12, 50 ,ug of denatured salmon
sperm DNA per ml), and incubated in the presence of 120 U
of S1 nuclease (Bethesda Research Laboratories, Inc., Gaithersburg, Md.) at 23°C for 70 min. Sl-resistant probe
fragments were resolved on 9 M urea-6% polyacrylamide
gels and visualized by autoradiography.
Quantification of gel autoradiographs. Autoradiographs
were quantified with a Zeineh soft-laser densitometer (model
SLR-504-XO; Biomed Instrument Co., Inc., Fullerton,
Calif.).
Analysis of polysome distributions. Single 100-mm-diameter dishes of transfected L cells, at just-subconfluent
density, were used for analysis of polysome distributions.
Polysome profiles were obtained by the method of Geyer et
al. (17). Each 100-mm-diameter dish of cells was washed two
times with 5 ml of ice-cold phosphate-buffered saline, then
lysed by the addition of 200 RI of polysome lysis buffer (10
mM Tris [pH 8.4], 10 mM NaCl, 3 mM MgCl2, 0.5% [wt/vol]
Nonidet P-40). The lysates were transferred to 1.5-ml Eppendorf tubes and vortexed for 10 s. Nuclei were pelleted,
and the resulting supematants were layered on 15 to 40%
(wt/vol) sucrose gradients in polysome gradient buffer (10
mM Tris [pH 8.4], 10 mM NaCl, 1.5 mM MgCl2). Gradients
were centrifuged at 32,500 rpm for 2 h at 4°C in a rotor
(SW41; Beckman Instruments, Inc.). Gradient fractions
were collected with a density gradient fractionator (model
185; ISCO, Inc., Lincoln, Nebr.) connected to a type 6
optical unit and a UA5 absorbance monitor. A total of 36
fractions of -0.4 ml each were collected in 1.5-ml Eppendorf
tubes containing 80 ,ud of 5 x proteinase K buffer (final
concentration, 200 p,g of proteinase K per ml). After all
fractions were collected, they were placed at 37°C for 1 h,
phenol-chloroform extracted, and then ethanol precipitated.
RNA from three consecutive fractions was pooled in a final
volume of 21 ,ud of diethylpyrocarbonate-treated water and
stored at -80°C.
RESULTS
Sufficiency of 1-tubulin codons 1 to 16 to confer j8-tubulin
autoregulation onto a chimeric mRNA. We have previously
shown (16) that insertion of a 106-base-pair segment of exon
1 of the chicken 132 tubulin gene (57 bases of 5'-untranslatedregion sequence and 49 coding nucleotides) into a chimeric
gene composed of the mouse MT I promoter and an HSV tk
gene body is sufficient to bring the resultant mRNA under
tubulin autoregulatory control. Further, since the complete
B2 5' untranslated region could be deleted from the authentic
gene without disrupting regulation, we predicted that the
regulatory domain must reside within the coding portion of
exon 1.
To confirm this prediction, we constructed a chimeric
gene (MT-C16-tk) in which only the first 49 translated
nucleotides (encoding amino acids 1 to 16) from the chicken
132 tubulin gene were fused in the proper translational
reading frame to the HSV tk gene (Fig. 1A). Transcription of
this fusion gene was driven by the mouse MT promoter
segment (which also provided a 68-base 5' untranslated
region). Translation of the RNA transcribed from MT-C16-tk
should produce a fusion protein, the first 16 amino acids of
which are 1 tubulin and the remaining 278 amino acids of
which are tk (the first AG initiation codon was the 1-tubulin
AG).
NUCLEOTIDES SPECIFYING 13-TUBULIN AUTOREGULATION
VOL. 8, 1988
ARI
TGA
ATG HaeI
1227
Rl
CB2
H3
RlI
t
A-G N'ut,
i+-4S-
+
--1
M3 ATO HaeINl
N®e/RI
N
+
+
_i
PI N de
GA
-
UI
n
MTtk
R
.GA
H SV-:_'xe
MT C16tk
18S M55 C16
18S M%35 Cp2 18S MP5 MTtk
B-- m
-+
-
--I+-+-+
622-
-
-
-+-+
__
-
527-
-_-
404-
-
M-+-+
+
m
9
309-
242-
217-
201-
1
2 3 4
9 10 11 12 13 1415 1617 18 19
FIG. 1. Sufficiency of 16 amino-terminal codons of chicken ,2 tubulin to confer tubulin autoregulation. (A) Schematic representation of
the cP2 gene (CP2) and hybrid genes MTtk and MT-C16-tk, (MTC16tk). Symbols:
, pBR322 sequences; -,,c2 or tk 5'-flanking,
, exon sequences corresponding to 5' and
3'-flanking, or intron sequences; I=, exon (Ex) sequences corresponding to coding regions;
3' untranslated regions; _, MT promoter, 5'-flanking and 5' untranslated sequences. RI, EcoRI site; H3, HindIII site; ATG, ATG
translation initiation codon; TGA, translation termination codon. (B) Mouse Ltk- cells transfected with cP2 (lanes 1 to 6), MTtk (lanes 7 to
12), or MT-C16-tk (lanes 14 to 19). Equal amounts of cytoplasmic RNA from control (lanes marked -) or colchicine-treated (lanes marked
+) cells were analyzed by S1 nuclease for 18S rRNA (18 S; lanes 1, 2, 7, 8, 14, and 15), mP5 mRNA (M,B5; lanes 3, 4, 9, 10, 16, and 17), c52
mRNAs (C12; lanes 5 and 6), MTtk (lanes 11 and 12), or MT-C16-tk (C16; lanes 18 and 19). Nucleotide size markers (in bases, lane 13) are
shown at the left. Arrows mark expected positions for fragments protected by cP2 and MT-C16-tk RNAs.
5 6 7 8
MT-C16-tk, MTtk (a control MT-tk gene that did not
contain any tubulin sequences but did retain the tk translation initiation codon), and the authentic c,2 tubulin gene
(Fig. 1A) were each transfected into duplicate dishes of
mouse L cells. S1 nuclease analysis was used to measure the
level of each transcript in RNA from control cells and from
cells to which colchicine had been added to induce microtu-
bule depolymerization and elevate the concentration of
unpolymerized tubulin subunits. Figure 1B shows the results
of S1 protection assays used to determine the levels of
endogenous mouse mP5 3-tubulin mRNA and the mRNAs
from the transfected genes. As previously reported (16),
RNAs from the authentic c32 gene were destabilized after
the elevation of the unpolymerized tubulin subunit content
1228
MOL. CELL. BIOL.
YEN ET AL.
A
RI
H
4
-
3
TGA
ATG
-.
t
-
S V -t
~~~~~H
k
_
Asr G Ie G:y Ala
Met Arg GM Hte Cal MIis Gle 1 Ala Gv :
Hl18 ATG AGC *AA C -G GAC A GC,CAG CC +C-7CtjG CCr7rr,CC .MC GAG- AC 'GMT CGCC A
1116 AT
AM GA.A AC G
C16 A.G
. C.-AG, AC M7
D: CC C--C M
CAC AC GAG -CC.CA GM.
GA
AC GA G CC C
C-C iA:
Y16 ACI AGA A.A AC AC- CAT ATC CCC GCA C-CC
Ser
.Lie
MB5
622527-
B
C
-A.-:
7,-:r
GAG AC- C
_ r CA
.
AACC-
GAA
A7 7,
18S
..4011im.
M16
Awa-1w-
-217
-201
404-
-190
U.mw
622-
527-
-180
4iW_i
.H18
-217
-201
404-
-190
.*
..180
:-
-180
622-D
527-
__Y16
-217
-201
(Fig. 1B, lanes 5 and 6), as were m,5 RNAs (Fig. 1B, lanes
3 and 4). Transfection of MTtk yielded RNA transcripts that
were completely insensitive to tubulin subunit concentration
(Fig. 1B, lanes 11 and 12). Incorporation of the first 16
codons of P tubulin as the first translated nucleotides of a
chimeric tk gene, however, yielded an RNA whose stability
was sensitive to the level of free tubulin subunits (Fig. 1B,
lanes 18 and 19). (The faint larger species seen in these lanes
correspond to minor transcriptional initiation sites that were
also evident in all RNAs expressed from the MT promoter.)
Quantitation of these results by densitometry confirmed
these qualitative impressions and revealed a 6.5-fold lower
level of MT-C16-tk RNAs in colchicine-treated cells (compared with a 9-fold loss of the RNAs encoding endogenous
mP5 tubulin). (In all cases, 18S rRNA levels were constant
[Fig. 1B, lanes 1, 2, 7, 8, 14, and 15], thereby insuring that
we had analyzed comparable amounts of RNA from control
and colchicine-treated cells.)
We conclude that a sequence that identifies the cP2
mRNA as a substrate for autoregulated changes in stability
resides within the first 16 translated codons.
1-tubulin autoregulatory signal is functionally conserved
during evolution. Since tubulin gene sequences are evolutionarily conserved (-65% nucleotide identity between human and yeast P tubulins; 12) and since tubulin autoregulation has been documented in a wide variety of species (see
Discussion in reference 33), we next tested whether sequences isolated from the amino-terminal-coding domain of
other P-tubulin genes also confer tubulin autoregulatory
properties when linked to nontubulin genes. For this, we
prepared hybrid genes analogous to MT-C16-tk but in which
the chicken tubulin sequence for codons 1 to 16 was substituted with codons 1 to 16 of the mouse mP5 gene, codons 1
to 18 of the human hp1 gene, or codons 1 to 16 of the S.
cerevisiae TUB2 P-tubulin gene (Fig. 2A). Each of the
hybrid genes was transfected into L cells, and the accumulated levels of transcripts were assayed by Si nuclease
analysis in RNA from control cells and from cells to which
colchicine had been added to elevate the level of free tubulin
subunits. Insertion of the mouse sequences (Fig. 2B), human
sequences (Fig. 2C), or yeast sequences (Fig. 2D) conferred
autoregulation onto an otherwise unregulated tk mRNA.
Moreover, addition of the amino-terminal segment from
each species had approximately the same quantitative effect
(6.5- to 8-fold).
Autoregulation of P-tubulin mRNAs is specified by as few as
13 nucleotides which encode the NH2-terminal four amino
acids. Inspection of the nucleotide and amino acid sequences
of the amino-terminal-coding sequences of hpl, mP5, cP2,
and yeast TUB2 genes (Fig. 2A) reveals a high degree of
sequence identity. At the nucleotide level, 32 of the 48 bases
404-
-190
the 16 amino-terminal codons of the mouse
-180
FIG. 2. Functional conservation of sequences sufficient to confer
P-tubulin autoregulation. (A) Schematic representation of hybrid
genes consisting of an MT promoter and 5' untranslated region, 16 to
18 codons for the amino terminus of ( tubulin, and 278 codons of tk
plus the tk 3'-flanking region. See the legend to Fig. 1 for explanation of symbols. Below the gene are shown the nucleotide and
corresponding amino acid sequences for the j-tubulin portions.
H18, MT-H18-tk, a hybrid gene containing 18 codons of the
human hpl gene; M16, MT-M16-tk, a hybrid gene containing
mP5
gene; C16, MT-
C16-tk, a hybrid gene which contains 16 codons of the chicken cP2
gene; Y16, MT-Y16-tk, a hybrid gene containing 16 codons of the S.
cerevisiae (-tubulin gene. (B to D) Mouse Ltk- cells transfected
with hybrid genes MT-M16-tk (B), MT-H18-tk (C), or MT-Y16-tk
(D). Equal amounts of cytoplasmic RNA from control (lanes marked
-) and colchicine-treated (lanes marked +) cells were analyzed by
S1 nuclease methods for endogenous m,B5 mRNAs (Mf5), 18S
rRNA, (18S), and MT-M16-tk, MT-H18-tk, or MT-Y16-tk RNAs. In
each panel, an arrow marks the nuclease-resistant fragment corresponding to RNAs initiated at the major MT transcription initiation
site. Nucleotide size markers (in bases) are shown at either side.
NUCLEOTIDES SPECIFYING j-TUBULIN AUTOREGULATION
VOL. 8, 1988
TGA
H3 ATG
ARI
-
Me- Arg
116 ATG AGG
M12 ATG AGG
N9 ATG AGG
N5 ATG AGG
YS ATG AGA
M4 ATG AGO
N2 ATG AGO
18S
H
SSV-tk
RilNde
I
L
Giu Ile Val His le Gin Aia Giy Gin Gys Giy An Gin Ie
GAA ATC GTG CAC ATC CAG GCGC GGA CAG TGT GGC MC CAG ATC
GM ATC GTG CAC ATC GAG GCC WGA CAG TOT G
GM
GAA
GMA
GAA
G
ATC
ATC
ATC
ATC
1229
G
GTG CAC ATG CAG GCC G
GTG C
ATT C
G
1BS
MB5
MB5
M12
622-
r-1
527-
M9
-
-201
-190
_
-180
-190
-180
404-
;
B
-160
C
-160
-ft
2X4
Y5
M5
-180
-160
-160
-i47
D
-
E
a--180
M4A
M4
_
W
-147
_
M2
-160
-147
-147
as
.-
F
G
-123
FIG. 3. Determination of the minimal sequence that can confer j-tubulin autoregulation. (A) Schematic representation of hybrid genes
corisisting of an MT promoter and 5' untranslated region, 2 to 16 codons of the amino-terminal-coding sequences for mP5 or yeast p tubulin
linked in the proper translational reading frame to 278 codons of tk, and the tk 3'-flanking region. See the legend to Fig. 1 for explanation of
symbols. Below the gene are shown the nucleotide sequences for the P-tubulin portions contained within genes MT-M16-tk (M16), MT-M12-tk
(M12), MT-M9-tk (M9), MT-M5-tk (M5), MT-Y5-tk (Y5), MT-M4-tk (M4), and MT-M2-tk (M2). (B to G) Mouse Ltk- cells transfected with
hybrid genes. Equal amounts of cytoplasmic RNA from control (lanes marked -) and colchicine-treated (lanes marked +) cells were analyzed
by S1 nuclease methods for endogenous mP5 mRNAs (M,5) and 18S rRNA (18S) and MT-M12-tk (B), MT-M9-tk (C), MT-M5-tk (D),
MT-Y5-tk (E), MT-M4-tk (F), or MT-M2-tk (G) RNAs. In each panel, an arrow marks the nuclease-resistant fragment corresponding to RNAs
initiated at the major MT transcription initiation site. Nucleotide size markers (in bases) are shown at either side (positions of markers for 622,
527, and 404 bases are denoted by dashes to the left of panels C to G).
encoding residues 1 to 16 are conserved throughout all four
genes. At the amino acid level, the encoded polypeptides are
identical among the vertebrates and only 3 substitutions
appear within the first 16 codons of the yeast sequence. To
determine more precisely which of these sequences was
sufficient to confer p-tubulin autoregulation, a series of
chimeric MT-tubulin-tk genes was constructed which contained the same MT and tk segments but which carried
between 16 and 2 P-tubulin codons at the amino terminus
(see Materials and Methods for details). Appropriate deletions were ligated in the proper translation reading frame to
produce a fusion protein composed of a few amino-terminal
amino acids of f tubulin followed by the majority of the
sequences for tk.
Figure 3A shows the sequences contained in several
fusion genes carrying m,5 sequences and one containing
yeast ,-tubulin sequences. Each of these genes was transfected and tested for whether its RNA transcripts were still
subject to P-tubulin autoregulation. When unpolymerized
tubulin subunit levels were elevated with colchicine, RNAs
transcribed from genes containing only 12 or 9 m[5 codons
(MT-M12-tk or MT-M9-tk, respectively) were destabilized
as if they were authentic 1-tubulin mRNAs (Fig. 3B and C).
(The heterogeneity in size of MT-M9-tk-protected fragments
1230
MOL. CELL. BIOL.
YEN ET AL.
A.-:,
m!
RI
-;A
13_C)1
C
C62
Met A-, Glu C1e Val His 7-e Gln Ala Gly Gln Cys Gly Asn Gln Ile Gly Ala Lys
A.(
TG
:C
Ci;;:
A
C"v-"'3V.
GA .;-C
C.C rAA C CkG GCC GC CAG ILIh;AC
A.
, J~u
-,'.7G
GCAG TGCG
-
A
CGAG
GC
MAG
GGC G.C
GAG GGC
GGC AACGAAC
ATC
GCr
GGCGGOGGG
GAGC
GGGCTG
- Gr AIG GCC GC;6 GCAG T GG0CC AAC AG ATC GGC GCT AAG
C.__
2O A-7CGO
C;-A
2
C f3
18S
MB5
CB2
-+ _-+-_+
-404
622-
_
527-
-309
B
18S
Ml35
_2
18S
622---_
622-
527-
M1n5+
3,4
+
-404
-404
-
527-
C
-309
-242
_
D
-
_-
.....-309
-.
-242
FIG. 4. Deletion of codon 2 or codons 3 and 4 disrupts autoregulation of RNAs from the cP2 tubulin gene. (A) Schematic diagram of genes
cP2A2 or cp2A3/4 for which site-directed mutagenesis was used to delete c,B2 codon 2 or condons 3 and 4, respectively. See the legend to Fig.
1 for explanation of symbols. (B to D) Mouse Ltk- cells transfected with c132 (B), c(32A2 (A2) (C), or c32A3/4 (A3/4) (D). Equal amounts of
cytoplasmic RNA from control (lanes marked -) and colchicine-treated (lanes marked +) cells were analyzed by S1 nuclease methods for
endogenous m,B5 mRNAs (M,5), 18S rRNA (18S), and the RNA from the transfected gene. Arrows mark the nuclease-resistant fragments.
Nucleotide size markers (in bases) are shown at either side.
was probably due to incomplete S1 digestion.) Genes containing only five mouse (MT-M5-tk) or yeast (MT-Y5-tk)
,B-tubulin codons were also regulated by the concentration of
tubulin subunits (Fig. 3D and E). Even a construct (MT-M4tk) with only the first four m15 codons was still regulated
efficiently (Fig. 3F), a qualitative result confirmed by densitometry. However, insertion of only two codons in front of
tk (MT-M2-tk) yielded RNAs that, like MTtk alone, were
insensitive to the tubulin subunit level (Fig. 3G). (In several
efforts, we failed to obtain a construct with only three
,B-tubulin codons.)
Nucleotides encoding amino acids 1 to 4 of ,B tubulin
essential for tubulin autoregulation. Although the 13 nucleotides which encode the first four amino acids of 1 tubulin are
sufficient to confer autoregulation onto heterologous mRNAs,
it remained possible that multiple, redundant regulatory
domains were present in an authentic gene. To determine
whether the codons for residues 1 to 4 were necessary and
sufficient to specify autoregulation, codon 2 or codons 3 and
4 were deleted from the authentic cP2 gene by site-directed
mutagenesis. The resultant genes (cp2A2 and c32A3/4; Fig.
4A) were transfected into L cells to test whether RNAs from
these genes were still substrates for autoregulation. (The
translation reading frame was unchanged in these deletions,
so that except for the absence of amino acid 2 or amino acids
3 and 4, an authentic 13-tubulin polypeptide would be translated.) Although RNAs from transfections of the authentic
cP2 gene yielded autoregulated instability as expected, the
absence of only codon 2 or codons 3 and 4 resulted in
complete insensitivity to autoregulation (Fig. 4B to D).
(Since the Si probe used was from the unmutated cP2 gene,
two fragments were detected in analyses of RNAs from
cp2A2 and c232A3/4. One fragment corresponded to S1
cleavage at the site of the deletion, whereas the larger
fragment [representing probe protection to the normal cP2
transcription initiation site] was due to incomplete S1 digestion at the site of the deletion.)
Since these deletions disrupted autoregulation, we conclude that the sequences within the first 13 translated nucleotides of (3 tubulin that are sufficient to confer autoregulation
are also necessary for specifying an RNA as a substrate for
autoregulation.
RNAs carrying the 13-nucleotide sequence must be translated beyond codon 41 to be substrates for autoregulation.
Previously, we have shown that insertion of a premature
translation termination codon at position 27 of the authentic
human tubulin gene M40 results in disruption of autoregulation (33). Further, inhibitors of protein synthesis that disrupt
NUCLEOTIDES SPECIFYING
VOL. 8, 1988
ARI
H3 ATG
+
TGA
+
_t_H_S V- tk
N
0-TUBULIN AUTOREGULATION
1231
RI/Nde
V t"11
1
M16 in-frame
Eaa -tbulir.
.t1G
c8,. : '
>:_.
3
0 G-A
i
CrcCG
c
--
%
Total 296..
817 out-of-frame
+
17 aa -:rub.-a..i..i XM
ATS (N;f 5 x-CC GAC -CT CGSA -
.
H-.S't k
GA- -
-
Totasl 41as
Y16 out-of-frame
Ml3
lSaa rTuIir.n
.rcG NN )t, 4
S
:9s7.tk
*
AC: C rcA
--A.
-
To'otel 31aa
H18 out-of-frame
'iaa Tubtlir. t MI3
ccA
.rc
.1146 or C-"C CC
18S
ii S.t kS
CGA
Total 33aa
18S
M17out
MB5
d+
622- e _-
MB5
+
Y16out
-+
+- -
527-217
0
404-
-217
-
-201
-201
-190
B
180
18S
MB5
-190
C
-180
H180ut
n
-217
Alr
-201
-190
-180
D
FIG. 5. Out-of-frame MT-tubulin-tk fusions whose RNAs encode truncated polypeptides are not substrates for tubulin autoregulation. (A)
Schematic diagram of MT-tubulin-tk genes in which the fusion between p-tubulin and tk coding sequences places the tk codons in either of
the two inappropriate translation reading frames. See the legend to Fig. 1 for explanation of symbols. aa, Amino acid. (B to D) Mouse Ltkcells transfected with MT-M17OUt-tk (M17,0ut; B), MT-Y160ut-tk (Y160ut; C), or MT-Hl80ut-tk (Hl80.u; D). Equal amounts of cytoplasmic RNA
from control (lanes marked -) and colchicine-treated (lanes marked +) cells were analyzed by S1 nuclease methods for endogenous mP5
mRNAs (M35), 18S rRNA (18S), and the RNA from the transfected gene. In each panel, an arrow marks the nuclease-resistant fragment
corresponding to RNAs initiated at the major MT transcription initiation site. Nucleotide size markers (in bases) are shown at either side. The
positions of markers for 622, 527, and 404 bases are indicated by dashes to the left of panels C and D.
polyribosomes unlinked tubulin RNA levels from the level of
free subunits, whereas a protein synthesis inhibitor (cycloheximide) that freezes mRNAs onto polyribosomes enhanced autoregulated tubulin RNA instability. Together,
these findings led us to propose that only RNAs that are
attached to polyribosomes are substrates for autoregulation.
If this is correct, then insertion of the amino-terminal codons
of P tubulin in front of tk should yield autoregulated RNAs
only when the tubulin and tk sequences are linked to
produce a long, open translation reading frame that allows
many ribosomes to be attached simultaneously to a single
RNA. In screening the MT-H-tk, MT-M-tk, and MT-Y-tk
deletion series (Fig. 2 and 3), we obtained numerous constructs containing various numbers of tubulin codons that
1232
YEN ET AL.
MOL. CELL. BIOL.
.
e
*m12^t
m__
.m35
- i
u
.-rnl2jt,,
CHX- CLC- +
+ +
- +
18S
C
18S
M135
M152out we"
M12
O
M12out
M12
s
1 2
3 4
FIG. 6. RNAs from out-of-frame MT-tubulin-tk genes were effi-
were linked to either of the two incorrect translational
reading frames of tk. Since in either of these inappropriate tk
frames translation termination codons are found 13 or 22
amino acids into the tk sequences, these MT-tubulin-tk
fusion genes can only encode truncated polypeptides.
Three such out-of-frame constructs that contained 16 to 18
codons of human, mouse, or yeast 1 tubulin are diagrammed
in Fig. 5A. The mouse construct (MT-M17OUt-tk) contained
precisely the same sequences as MT-M16-tk (Fig. 2), except
that it possessed two additional tubulin nucleotides. The
translation product consisted of 17 amino acids of 1B tubulin,
2 amino acids encoded by linker sequences, and 22 amino
acids produced from one of the two wrong reading frames of
tk. The human or yeast construct (MT-H18Out-tk or MTY160ut-tk, respectively) yielded a polypeptide composed of
18 or 16 residues of 1B tubulin, respectively, 2 linker amino
acids, and 13 residues translated from the other incorrect tk
frame.
When these out-of-frame constructs were transfected and
tested for autoregulation, the stabilities of all encoded RNAs
were independent of the free tubulin concentration (Fig. 5B
to D), despite the fact that RNAs from the corresponding
in-frame fusions were autoregulated (Fig. 2B to D). Thus,
RNAs that contain the identifier sequences are not substrates for autoregulation when their translation terminates
before codon 42.
RNAs from out-of-frame MT-tubulin-tk genes fail to autoregulate not because of failure to be translated. Since none of
the RNAs transcribed from the out-of-frame MT-tubulin-tk
genes were autoregulated even though they encoded polypeptides of up to 41 amino acids, the most likely interpretation was that translation must proceed 3' to codon 41 for
autoregulation to occur. However, it was also possible that
the out-of-frame RNAs failed to regulate because they were
not translated efficiently (and thus were not present at the
site at which autoregulation takes place). To test this hypothesis, we analyzed polysome profiles of RNAs from cells
transfected with MT-M12,Ut-tk, both before and after a
colchicine-induced increase in the free tubulin subunit concentration (Fig. 6A). When each gradient fraction was analyzed for MT-M12OUt-tk RNA or for the endogenous mouse
m15 RNA, most of the out-of-frame MT-M12out-tk RNA
cosedimented with di- and trisomes. Since a ribosome apparently spans -15 to 20 codons (34), this is the position
predicted for an mRNA that efficiently attached to ribosomes but contained only 36 codons. A parallel analysis was
also performed on RNA from cells transfected with the
analogous in-frame MT-M12-tk gene (Fig. 6B). Like m,B5
RNAs, MT-M12-tk RNAs were found in the heavy polysome
fractions, and these polyribosomal MT-M12-tk RNAs were
degraded in cells with elevated free tubulin subunit concentrations (Fig. 6A and B, rows marked +). No corresponding
loss of polysomal MT-M12ou,-tk RNAs was observable after
ciently translated but were not substrates for autoregulation even
when ribosomal dwell time was increased with cycloheximide. (A)
Polysome profiles generated from lysates ofjust-subconfluent dishes
of L cells transfected with MT-M12OUt-tk, with or without colchicine
treatment to elevate the concentration of free subunits. The profile
shown is from cells not treated with colchicine (although the two
profiles were essentially indistinguishable; 33). The direction of
sedimentation (big arrows) is left (top of gradient) to right (bottom of
gradient). The small arrows mark the positions of the 40S, 60S, and
80S (monosome) ribosomal subunits. The positions of disome (2'),
trisome (3'), etc. are marked with numbers. Arrowheads (V)
delineate the portions of both gradients from which fractions were
collected. The rows beneath the profile show Si analysis of RNA
recovered from profile fractions. m12,ut, RNAs from MT-M12OUt-tk;
mP5, RNAs from mP5; -, RNA from control cells; +, RNA from
colchicine-treated cells. (B) Results of an experiment analogous to
that described in the legend to panel A, except that cells were
transfected with the in-frame gene MT-M12-tk. m12j., RNA from
MT-M12-tk. (C) Four parallel dishes of L cells transfected with
MT-M12OUt-tk or MT-M12-tk. At 4 h before harvest, no drug,
colchicine (CLC), cycloheximide (CHX), or colchicine plus cycloheximide was added (to 10 ,uM for colchicine and to 5 ,uM for
cycloheximide). Cytoplasmic RNA was prepared and analyzed by
S1 nuclease for 18S rRNA (18S), endogenous mP5 RNAs (M15),
and MT-M12OUt-tk (M12Out) or MT-M12-tk RNAs (M12). -, Absence of drug; +, presence of drug.
VOL. 8, 1988
NUCLEOTIDES SPECIFYING P-TUBULIN AUTOREGULATION
treatment with colchicine. We conclude that MT-M12out-tk
RNAs are efficient substrates for translation, despite their
inability to autoregulate.
Failure of RNAs from out-of-frame MT-tubulin-tk genes to
autoregulate not due to diminished dwell time while attached
to ribosomes. One obvious difference between substrate
(in-frame) RNAs and nonsubstrate (out-of-frame) RNAs is
the time that a ribosome remains attached to an individual
RNA. Since the initial RNA degradation step occurs preferentially on ribosome-bound RNAs (33; see also above), a
possible explanation for the failure of out-of frame RNAs to
autoregulate is a kinetic one: ribosomes may elongate and
release faster than the recognition-cleavage step. We tested
this by treating cells transfected with MT-Ml20ut-tk with
cycloheximide, an inhibitor that blocks translation elongation but does not affect translation initiation (39). Cycloheximide thus traps mRNAs onto ribosomes and drives almost
all ribosomal subunits into polyribosomes (14, 20, 33). Moreover, as might be anticipated, cycloheximide enhances tubulin autoregulation in L cells for either endogenous mP5
RNAs or in-frame MT-tubulin-tk fusion genes even in the
absence of complete microtubule depolymerization (Fig. 6C,
compare lanes 1 and 3). Nonetheless, the very significant
increase in ribosome dwell time induced by cycloheximide
failed to restore autoregulation to MT-M120Ut-tk RNAs.
DISCUSSION
We have now identified the first 13 translated nucleotides
as the sequences necessary and sufficient to specify a
ribosome-bound mRNA as a substrate for P-tubulin autoregulation. From earlier observations with protein synthesis
inhibitors, we concluded that the primary substrates for
regulated mRNA instability were polyribosome-bound
RNAs (33). We have now found that chimeric RNAs that
encode truncated translation products are not substrates for
autoregulation. Although precisely how much farther translation must proceed to restore autoregulation remains to be
elucidated, RNAs subject to autoregulation must be translated somewhere beyond codon 41. Polysome profiles and
the use of cycloheximide to slow translocation of ribosomes
have shown that the failure of out-of-frame RNAs to regulate
is not simply due to inefficient association with polysomes
nor to the shorter time an inRNA is associated with any one
ribosome.
Consideration of all of these data suggests two models for
the novel mechanism that establishes the regulated instability of polyribosome-bound P-tubulin RNAs. The first possibility is that the recognition event that marks an RNA as a
substrate is the nucleotide sequence of the RNA itself. As
the level of unpolymerized subunits is increased, the tubulin
subunits bind (directly or indirectly) to the amino-terminalcoding sequences of a translating 1-tubulin RNA. Since the
ribosome may cover -50 nucleotides of the mRNA (34),
presumably the RNA recognition sequence would be accessible for binding only immediately after it emerges from the
ribosome (and before it reinitiates on a second ribosome).
The binding event must induce a change in the conformation
of the tubulin mRNA which makes it a more efficient
substrate for a preexisting ribonuclease (e.g., by inhibiting
its refolding into a native ribonucleoprotein). A problem with
this model is that it is not obvious how subunit binding to the
RNA could be enhanced on mRNAs saturated with cycloheximide-stalled polyribosomes.
The alternative model exploits the observation that the
recognition sequence encodes a four-residue, amino-ter-
1233
minal polypeptide. Since a- and 1-tubulin subunits interact
with each other (Kd = 10-8; 13), the true recognition event
could be a protein-protein interaction in which free subunits
interact with the nascent tubulin polypeptide immediately as
it emerges from the large ribosomal subunit. The proteinprotein binding event would then stimulate tubulin mRNA
degradation, either by activation of a ribosome-associated
nuclease or by induction of transient ribosome stalling that
leaves tubulin RNAs in a more exposed conformation.
Precisely how a protein-protein binding event could be
transduced through a ribosome to yield RNA cleavage is not
clear, although strong precedent for protein-protein binding
inducing ribosome stalling is provided by the proposed role
of a signal recognition particle in the translation of secreted
proteins (42). This peptide recognition model can easily
account for the finding that only mRNAs attached to ribosomes are substrates for autoregulated RNA instability.
Obviously, linkage of enhanced tubulin RNA instability to
subunit binding of the newly made polypeptide can only be
achieved while the mRNA and polypeptide are physically
linked, i.e., while both are still held on the ribosome.
Further, since 30 to 40 amino acids of the newly translated
polypeptide are covered by the large ribosomal subunit (43),
this model can also easily explain why RNAs that yield
truncated translation products are not substrates for autoregulation. In these cases, RNA release from the ribosome
precedes the emergence of the 3-tubulin amino-terminal
peptide.
Although we have not proven which of these two alternatives is correct, several considerations make the second
model more attractive. First, the encoded polypeptide (MetArg-Glu-Ile) is absolutely conserved among all Pi tubulins
sequenced to date, whereas a significant number of base
substitutions within the 13-nucleotide recognition domain
have been tolerated during evolution for example, see Fig.
2A. Moreover, a search of the Los Alamos DNA sequence
library revealed that the RNA sequence most conserved
evolutionarily (AUG NGN GAG/A AUC G/A) is not unique
to 3-tubulin RNAs, although a similar search of the protein
sequence data base revealed that Met-Arg-Glu-Ile is found
exclusively in 13 tubulin. Even with the possibility that the
true minimal sequence may be only Met-Arg-Glu (we were
unsuccessful in isolating a fusion construct that carried only
the first three 3-tubulin codons), our search of the protein
library revealed that while several known proteins carry this
sequence internally, only tubulins (both 13 and a) have it at
their amino termini. In any event, future site-directed mutants in which the recognition sequences have been frameshifted into an untranslated reading frame will be needed to
unambiguously distinguish between these two models.
The identification of the sequence that specifies 3-tubulin
mRNAs as substrates for autoregulated instability further
highlights the unanswered question of the functional end to
which autoregulation is utilized. The recognition that autoregulation has been widely conserved during speciation (see
Discussion in reference 33) strongly suggests that it serves
an important functional role. At first glance, autoregulation
appears to be largely redundant, since the polymerization
reaction in vitro is self buffering: subunits continue to
assemble until the pool of subunits is depleted to the critical
concentration at which free subunits are at steady state with
polymer. However, Mitchison and Kirschner (T. J. Mitchison and M. W. Kirschner, Cell Biophys., in press) have
recently demonstrated that the concept of a unique critical
concentration for a microtubule array in a closed system
(i.e., in a cell with a fixed amount of tubulin and whose
1234
MOL. CELL. BIOL.
YEN ET AL.
microtubules are nucleated from specific centrosomal sites)
is not meaningful. Rather, microtubules can polymerize at
almost any subunit concentration provided that nucleation is
sufficiently rapid. In light of this, the number and stability of
nucleated microtubules depend on two factors: nucleation
capacity and subunit concentration. Thus, as we initially
proposed (33), autoregulation of tubulin synthesis may represent the cellular mechanism that establishes a level of
unpolymerized subunits necessary to ensure a nucleated
array of dynamic microtubules.
Finally, in addition to the present example of ,B tubulin,
the expression of an increasing number of eucaryotic genes
has now been identified to be regulated at least in part by
RNA stability. For example, a series of lymphokine, cytokine, and proto-oncogene mRNAs contain within their 3'
untranslated regions a 50-base AU-rich sequence that destabilizes the RNA (37). Similarly, histone mRNAs carry specific sequences in their 3' untranslated regions that confer
cell cycle-dependent RNA stability (4, 19). Moreover, the
pathways that establish RNA stability for all of these examples are similar insofar as RNA instability appears to require
active translation (e.g., for RNAs encoding c-myc [27], c-fos
[22, 30, 31], and histones [2, 19, 38]). However, unlike the
case for tubulin in which cycloheximide-induced ribosome
stalling enhances mRNA instability (33), the instability of
these other RNAs appears obligatorily linked to ongoing
protein synthesis. For these RNAs, translational arrest with
cycloheximide results in increased stability of the corresponding RNAs, a feature that has led to the suggestion that
mRNA degradation is coupled to the completion of translation (27). In this regard, it has been shown that histone
mRNA degradation not only requires the presence of histone
mRNAs on polysomes (2) but, in addition, that translation
proceeds close to the 3' of the mRNA (19).
Despite differences in detailed mechanisms, the present
examples of regulated mRNA instability leads us to propose
the existence of a common motif in which RNA degradation
is integrally linked to ribosome attachment and translation.
In light of this, we would also offer the appealing suggestion
that the RNase(s) that affects the cleavages may actually be
localized to actively translating ribosomes (and perhaps is a
peripheral subunit thereof).
ACKNOWLEDGMENTS
T.J.Y. and J.S.P. have been supported by postdoctoral fellowships from the American Cancer Society and the National Institutes
of Health (NIH), respectively. D.A.G. has been supported by an
NIH predoctoral training grant. This work has been supported by a
grant from the NIH to D.W.C., who is also the recipient of an NIH
research career development award.
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