Polyadenylation and U7 snRNP-mediated cleavage

Polyadenylation and U7 snRNP-mediated cleavage: alternative modes of RNA 3' processing in two avian histone HI genes

A n d r e w L. Kirsh, 1,2,4 Mark Groudine , 1,3 and Peter B. Chal loner I

XDivision of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104 USA; Departments of 2Pathology and aRadiation Oncology, University of Washington School of Medicine, Seattle, Washington 98195 USA

The six chicken histone HI genes have T-processing sequences typical of replication-dependent histone genes, which are expressed as poly(A)- mRNAs. However, by Northern analysis of RNA from several adult chicken tissues, as well as from embryonal skeletal muscle in vivo and in vitro, we have observed histone HI transcripts longer than those predicted on the basis of the published genomic sequences. These RNAs are polyadenylated transcripts of the genes H1.01 and HI.10, which encode the 'c fraction' HI protein subtypes. Both transcripts contain an internal stem-loop and purine-rich box associated with the 3' processing of poly(A)- histone mRNAs. The 2-kb poly(A) + HI.01 transcript is present at high steady-state levels in tissues with low rates of DNA synthesis, has a longer half-life than the poly(A)- mRNA from the same gene, and is polyribosomal in embryonal skeletal muscle. The 1-kb poly{A) + HI.10 RNA is the major HI.10 transcript in adult skeletal muscle. The properties of these RNAs suggest that they may contribute to the relaxed replication dependence of c fraction subtype expression. The polyadenylation signals of both genes are unusual in their association with processed [nonhistone) pseudogene-like elements, an arrangement with possible implications for the mechanism of alternative 3'-end formation in these genes.

[Key Words: Polyadenylation; histone HI]

Received August 8, 1989; revised version accepted October 5, 1989.

The majority of the histone genes of higher eukaryotes can be grouped into two classes: replication-dependent histones and replacement variants (Zweidler 1984). Rep- lication-dependent histones constitute the predominant class and are expressed primarily in S phase of the mitotic cell cycle. During S phase, transcriptional initiation, mRNA stability, and possibly processing of precursor RNA to form the mature nonpolyadenylated 3' end are maximal, resulting in a 15- to 50-fold increase in histone mRNA levels (for recent reviews, see Birnstiel and Schaufele 1988; Marzluff and Pandey 1988; Mowry and Steitz 1988). The sequence elements that are necessary for each of these modes of regulation and some of the trans-acting factors that recognize these elements have been identified. Histone type-specific promoter elements and associated factors act to stimulate transcriptional initiation by three- to fivefold in S phase (Heintz et al. 1983; Sittman et al. 1983; Fletcher et al. 1987; Dalton and Wells 1988a, b). The majority of the S-phase increase in histone mRNA concentration is due to en- hanced stability (Heintz et al. 1983; Sittman et al. 1983). In the pre-mRNAs of replication-dependent histones of

4Corresponding author.

all species, a short 3'-nontranslated region is followed by two sequence elements that flank the mature 3' end: a highly conserved element - 23 nucleotides long, including a region of hyphenated dyad symmetry, which forms a 6-base stem and a 4-base loop, and a closely associated purine-rich sequence of - 10 nucleotides. Matu- ration of the 3' end occurs by endonucleolytic cleavage several bases downstream of the s tem-loop and yields a mRNA with a terminal ACC(C)A (Birchmeier et al. 1984; Krieg and Melton 1984). Accurate cleavage both in vivo and in vitro requires the purine-rich box (Georgiev and Bimstiel 1985; Luscher et al. 1985; Vasserot et al. 1989). The s tem-loop increases the efficiency of cleavage (Mowry et al. 1989; Vasserot et al. 1989) and binds an Sm-precipitable factor in nuclear extracts (Vas- serot et al. 1989). These two elements with - 6 0 bases of contiguous nonconserved downstream RNA are suffi- cient to act as a substrate in the cleavage reaction (Stauber et al. 1986). The core of the purine-rich sequence forms a duplex with the complementary sequence in the U7 snRNA component of the U7 small nuclear ribonucleoprotein (snRNP) particle (Cotten et al. 1988; Gilmartin et al. 1988). This factor is present at a constant level throughout the cell cycle in at least one cell line (Luscher and Schumperli 1987). Mowry and

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Steitz (1987a, b) have detected a factor in HeLa cell nuclear extracts that binds to the s tem-loop of exogenous precursor histone RNA. In addition, a heat-labile cleavage activity from both temperature-sensitive mutant mouse 21-Tb cells and mouse C127 fibroblasts has been described. This activity is depleted in extracts of temperature-arrested 21-Tb cells and serum-starved C127 cells (Luscher and Schumperli 1987; Stauber and Schumperli 1988). Whether a similar decrease in cleavage activity occurs in cycling cells outside of S phase is not known. At the end of S phase, the cytoplasmic histone mRNA is specifically degraded in the 3' to 5' direction through a translation-dependent mechanism that does not affect polyadenylated mRNAs (Sittman et al. 1983; Sive et al. 1984; Ross and Kobs 1986; Graves et al. 1987; Pelz and Ross 1987; and references therein).

In contrast to the replication-dependent histones, the expression of the replacement variant histone genes does not fluctuate as greatly throughout the cell cycle (Zweidler 1984). Replacement variant histone mRNAs are not cell cycle regulated because they lack the 3'-terminal s tem-loop characteristic of the replication-dependent RNAs and are polyadenylated. In addition, some replacement variant genes lack the promoter proximal sequences required for the S phase-specific increase in transcription. The replacement variant RNAs are found predominantly in quiescent and differentiated cells, where the histone subtypes they encode replace replication-dependent histones to varying degrees (Zweidler 1984).

In chickens, the HI genes H1.01 and HI.10 encode the 'c fraction ' protein subtypes H 1 c' and H 1 c, respectively (Shannon and Wells 1987). The presence of lysine-to-ar- ginine substitutions in these subtypes and their preva- lence in nondividing tissues have led to suggestions that they may have increased capacities to condense chro- matin (Coles et al. 1987) and may serve a function analo- gous to that of the mammalian protein H1 ° (Winter et al. 1985a). One or both of the c fraction proteins (which comigrate on most gel systems) are preferentially expressed in adult tissues with low rates of cellular proliferation (Berdnikov et al. 1976) and in embryonal myotubes both in vivo and in vitro (Winter et al. 1985a, b). The basis for the relaxed dependence of c fraction expression on DNA synthesis is unclear, as both H1.01 and HI.10 have promoter and 3'-processing sequence elements typical of replication-dependent histone genes (Sugarman et al. 1983; Coles and Wells 1985; Coles et al. 1987), and H1.01 expression is cell cycle regulated in HeLa cell transient expression assays (Dalton and Wells 1988a).

Here, we demonstrate that the H1.01 gene is expressed predominantly as a polyadenylated transcript in tissues with few dividing cells. In addition, the major HI. 10 gene transcript in adult skeletal muscle is polyadenylated. The sequences of cloned cDNAs reveal that each of these poly(A) + RNAs contains an internal s t e m - loop and purine-rich element. In both the H1.01 and HI. 10 genes, the AATAAA hexamer signaling polyaden-

Alternative RNA processing in avian histone H1 genes

ylation precedes a genomic poly(A) tract in a downstream element resembling a processed (nonhistone) pseudogene. The polyadenylated H1.01 transcript is associated with polyribosomes in embryonal muscle, and its stability suggests that these polyadenylated transcripts may have a role in the preferential synthesis of the H1 subtypes that they encode.

Results

Polyadenylated H1 transcripts

To examine H1 gene expression in tissues with different replication rates, we isolated RNA from various tissues of the adult chicken. Northern hybridization with the insert from pCH42, containing conserved Hi-coding sequence from the H1.01 gene, detected two major size classes of HI transcripts in these RNA preparations: RNAs of - 7 6 0 nucleotides corresponding to the predicted poly(A)- mRNAs from the six chicken H1 genes (Coles et al. 1987), and longer transcripts of - 2 kb (Fig. 1). In any tissue, the abundance of the 2-kb H1 RNA was inversely correlated with mitotic activity. The 2-kb transcript was the single most abundant HI RNA in car- diac muscle, skeletal muscle, and liver, all of which have few dividing cells and a high c fraction HI content (Berdnikov et al. 1976). It was also detected at lower levels in brain, lung, and spleen, and it is a minor species

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C Figure 1. Northem analysis of chicken tissue and cultured myotube RNAs. {A) Total RNA from adult tissues, probed with the pCH42 insert containing conserved HI-coding sequence. (B) Total RNA from 14-day embryonal breast muscle, cultured embryonal breast muscle 4 days postplating without cytosine arabinoside (Ara C), and cultured embryonal breast muscle at 6 days postplating with or without treatment with Ara C, probed with the H1.01 gene-specific oligonucleotide HI.01.3', complementary to the 3'-nontranslated region. (C) Poly(A)- and poly(A) + RNA from cultured emblyonal breast muscle 4 days postplating, without Ara C, probed with HI.01.3'. Fifteen micrograms of each RNA was loaded in each lane in A and B. Eighteen micrograms of poly(A)- and 3.5 ~g of poly(A) + RNA were loaded in C. Sizes of HiudIII-digested bacteriophage ), DNA markers are indicated at left. Sizes of RNAs estimated by comparison to DNA markers are at right.

GENES & DEVELOPMENT 2173




Kirsh et al.

in testis and in bone marrow, which has the highest rate of DNA synthesis of the tissues examined.

The high steady-state levels of the 2-kb RNA in tissues known to be enriched in c fraction H1 protein subtypes suggested that it may be expressed by the H1.01 or HI.10 gene. Northern blots of total cellular RNA from day-14 embryonal breast muscle and cultured embryonal myotubes (Fig. 1B) were probed with HI.01.3', an oligonucleotide complementary to unique sequence in the 3'-nontranslated region of the H1.01 gene. This probe hybridized to both 760-nucleotide and 2-kb transcripts, demonstrating that the H1.01 gene is expressed as two stable RNA species. In cultured muscle treated with cytosine arabinoside to eliminate dividing cells, the 2-kb RNA is the predominant HI.01 transcript (Fig. 1B), suggesting that alternative RNA processing is responsible for the expression of H1 c' protein in myotubes. The levels of 760-nucleotide and 2-kb RNAs in cultured embryonal muscle before myotube formation are similar (data not shown) to those in 4-day, fused cultures in the absence of cytosine arabinoside treatment (Fig. 1B).

The high steady-state levels of the 2-kb RNA in tissues composed predominantly of quiescent cells suggested that some structural feature may allow this RNA to escape the replication-dependent gene-specific degra- dative process. Because polyadenylated mRNAs are not degraded by this mechanism, we hypothesized that the H1.01 2-kb RNA may be polyadenylated. Probe HI.01.3', which is complementary to unique sequence in the 3'-nontranslated region of the H1.01 gene, was used for Northern analysis of poly(A)- and poly(A) + RNA fractions from cultured embryonal muscle (Fig. 1C). As expected, the 760-nucleotide mRNA is in the poly(A)- fraction. The 2-kb transcript is in the polyadenylated fraction, suggesting that alternative 3' processing of H1.01 precursor RNA occurs.

Polyribosome analysis and message stability

The observations discussed above led us to ask whether the H1.01 polyadenylated transcript is translated and whether an increase in the stability of the 2-kb RNA, possibly due to polyadenylation, may contribute to the preferential expression of H1.01 in certain tissues. We prepared polyribosomes from the breast muscle of 14- day chick embryos, treated half the material with puromycin to specifically release mRNAs from polyribosomes, and fractionated the two samples on sucrose gra- dients. Figure 2A shows the shift in Azs4 toward the top of the gradient loaded with the puromycin-treated sample, relative to the untreated sample, reflecting the dissociation of polyribosomes into free mRNA and subunits. Slot blots of RNA purified from the gradient fractions were probed with the insert of pCH42.8, a unique sequence probe that maps downstream of the normal cleavage site and hybridizes to the 2-kb, but not to the 760-nucleotide, RNA on Northern blots (data not shown) to detect the H1.01 2-kb transcript. The results are presented in Figure 2B. The peak of hybridization is

A.

B.

20 r t 1 .0 : Od

o

/ /

Control

+ Puromycin

1 2 3 4 5

Froction Number

Figure 2. (A) Sucrose gradient profiles of control (solid line) and puromycin-treated (dashed line) polyribosomes. (B) Filter hybridization of extracted RNA probed with the pCH42.8 insert, which hybridizes to the 2-kb but not the 760-nucleotide H1.01 RNA. Equal proportions of the 10 RNA samples were applied to the filter. The top of each gradient is to the left in A and B.

shifted from the polyribosomal region of the puromycin- free gradient toward the position of free RNA after puromycin treatment, indicating that this transcript is in polysomes and contributes to the cellular pool of subtype Hlc ' .

Then we measured the relative stabilities of the poly(A)- and poly(A) + H1.01 mRNAs through the cell cycle. Confluent cultures of chick embryo fibroblasts were synchronized by a 12-hr period of incubation in medium with reduced serum concentration, followed by serum stimulation. Cells were treated with actinomycin D for 0, 45, 90, and 135 min at 0 and 10 hr, following the increase in serum concentration. These time points cor- respond to Go and early S phase of the cell cycle, respectively (Thompson et al. 1985). Northern analysis of total cellular RNA probed with H1.01.3' showed that the half-life of the nonpolyadenylated mRNA is short, as is typical of replication-variant mRNAs. In contrast, the half-life of the 2-kb polyadenylated transcript is rela- tively long: The steady-state level of this mRNA was essentially constant for the course of the experiment (Fig. 3). Thus, polyadenylation of the H1.01 2-kb mRNA is correlated with increased stability. This suggests that alternative 3'-end formation allows H1.01 gene expression to become disengaged from the quantitatively most significant aspect of histone gene cell-cycle regulation.

cDNA sequence of the 2-kb HI.O1 transcript

To investigate the mechanism of 3'-end formation of the H1.01 transcripts and to determine whether the polyadenylated message contains histone-specific 3'-processing signals, we isolated cDNA clones of this transcript. We used pCH42.8 to screen a cDNA library derived from poly(A) + RNA from cultured chick embryo myotubes, identified 12 positive clones, and sequenced 1 of them (Fig. 4A). The sequence indicates that the 2-kb RNA is

2174 GENES & DEVELOPMENT




Ohr 10 hr 0 ~135' O ---- '-135'

Figure 3. Northern analysis of RNA from act inomycin D- treated, serum-st imulated chick embryo fibroblasts with probe H1.01.3. Lanes were loaded with RNA from cells at 0, 45, 90, and 135 min Cleft to right) of treatment wi th act inomycin D, after 0 or 10 hi of serum stimulation. We have not mapped or otherwise analyzed the minor RNA at 3.5 kb.

processed from a precursor RNA that is not cleaved at the usual site but is polyadenylated at a site ~790 nucleotides downstream, following an AAUAAA sequence. This yields a molecule with an internal s t e m - loop and purine-rich element {Fig. 4A, B). The AATAAA element is part of a processed pseudogene-like sequence that lies between H1.01 and the neighboring H2A gene, in the same orientation as H1.01. This element is similar to the chicken sequence described by Robins et al. (19861 in that it has no obvious promoter sequences, is flanked by direct repeats, has an open reading frame {nucleotides 1727-2044), a poly(A)addition signal (starting at position 2161), and a genomic poly(A) tract (nucleotides 2173-2194) but no parent copy (data not shown) in the genome. [Probes complementary to sequences up- stream of the H1.01 promoter and immediately downstream of the genomic poly(A) tract do not detect the 2-kb mRNA on Northern blots (data not shown). This provides further evidence that the 2-kb mRNA origi- nates from the H1.01 promoter and confirms that it has a poly(A) tail beginning at nucleotide 2173, that is, the eDNA we have sequenced was not primed by oligo{dT) from an RNA containing an internal poly(A) tract].

A polyadenylated H1.10 transcript

The sequence of the 2-kb H1.01 cDNA led us to examine the published 3'-flanking sequences of the remaining five chicken H1 genes to determine whether any contain sequences that may allow the expression of polyadenylated transcripts. We observed that the 3'-flanking sequence of HI.10 has a processed pseudogene-like element that could contribute a polyadenylation signal (Fig. 6A). Northern hybridization of total RNA from several adult tissues with the oligonucleotide probe H1.10.907 (derived from unique sequence in the 3'-nontranslated region of HI. 10) detected an ~ 1-kb transcript in skeletal muscle (Fig. 5). Northern hybridization of poly(A)- and poly(A) + RNA from cultured myotubes with the HI. 10- specific probe Hl.10.5' confirmed that the 1-kb HI.10


A. A-RICH G-RICH

~CAAACACA.AATCGAGCACACCGAAGGGCTCCCCGGCCGTGCAGCGGCGCGGGCTTAGC 5 6 0

CCAAT TATA AACGCACCAATCACCGCGCGGCTCCTCTCTAAAAATACGAGCATCTGACCCGCGCCAGCC 620

C..._~TTGTGTTCGCCTGCTCCGCAGAGGACTGCGCCGCGATGTCCGAGACCGCTCCCGCCG 6 8 0

CCGCCCCCGATGCGCCCGCGCCCGGCGCCAAGGCCGCCGCC&AG.,M~GCCGAAGAAGGCGG 740 CGGGCGGCGCCAA&GCCCCCAAGCCCGCGGGCCCCAGCGTCACCGAGCTGATCACC~GG 800 CCGTGTCCGCCTCCAAGGAGCGCAAGGGGCTCTCCCTCGCCGCGCTCAAGAAGGCGCTGG 8 6 0 CCGCCGGCGGCTACGACGTGGAGAAGAACAACAGCCGCATCAAGCTGGGGCTCAAGAGCC 920 TCGTCAGCAAGGGCACCCTGGTGCAGACCAAGGGCACCGGCGCCTCGGGCTCCTTTCGGC 980 TCAACAAGAAGCCGGGTGAGGTGAAGGAGAAGGCTCCGAGGAAGCGAGCGACTGCTGCCA 1 0 4 0 AGCCCAAGAAGCCGGCGGCCAAGAAGCGTGCGGCTGCTGCCAAGAAGCCCAAGAAGGCGG 1100 CGGCGGTCAAGA&GAGCCCCA&GAAAGCCA&GAAGCCGGCGGCTGCCGCCACCAAGAAGG 1 1 6 0 CGGCCAAGAGCCCCAAGAAGGCCGCCAAGGCTGGCCGCCCCAACAACGCCGCCAAGAGCC 1220 CGGCCAAGGCAA&GGCGGTGAAGCCCA&GGCTGCCAAGCCCAAGGCGACC~CCCAAGG 1280 CGGCCAAGGCCAAGAAGACGGCAGCCAAGAAGAAGTAAGTTATCCCAGAAGAGTCCTGCT 1340

HI DYAD E L ~ E N T ~ PURINE-RICH CTACCTATTTTGATATCCAACGGCTCTTTTAAGAGCCACCCACACTTTC¢~T~&AGGAGC 1400 TGAGGCACCGAGGTCGTCAGAAACTTCCAGCACGGAGGCAGCAATTCGTAAGTCGTCAGA 1 4 6 0

CGTCAATTGCCTTTTCCCCTCCGATTACCGAAACCTAACG~-G~TGAACGCGGCGG 1520

~R 9 C T T T A G G G A A G T G T A G A C T T T G T A T C T T T T C ~ G T T T G A C T A C C G T G A A G A 1580 AACGTTTTGTAATGATTTGATAAAAATCGGGTGACACTTTTTTTAAGAATATATTTTGTA 1640 ACAGAAGTAATGGATTTCCCAGGCGCAAGCTACTACTGAGCCATGTCTAACGTGTTGTGT 1 7 0 0 TGTTCCTCTTTAAGGTGTCTCCTT~TTTTGTGTATTAGGGGAAGACGGGAGATTT 1 7 6 0

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TTCTTACTGACGCCGTAACAGCCCCGAGCTCTCCCATCTCTTTTGTTCCCGCTGAGACAG 1820 L T D A V T A P S S P I S F V P A E T E

AACAGCGGCTTCTGCTGTTGGAA~GCCCGCCCTGGCCGAGGATTGGCCACGAGGAGCCC 1880 Q R L L L L E K P A L A E D W P R G A R

GGCCCGCTGCCCCTTCCCCCTCCCACCGCAGTCCCCGCCTTGGGCCCGGCGCTTTTGGCC 19~0 P A A P S P S H R S P R L G P G A F G R

GCGTTCAAGAAAGGAACAGGCGTGGGGGAAAGGAGGGGGGAGGGGGGCGGGCGCTGACGG 2000 V E E R N R R G G K E G G G G R A V T G

GACCTCCCGGAACGCATTGGTTTCCTTTCGTACGATATACGAAC~AGTGTAACGGCG 2 0 6 0 P P G T H W F P F V R Y T N

CGTCCCGGGAGAAACTTCTTTTGGGAGAACGCTTTG~TGTTAACGGAAGCA 2120

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Figure 4. (A) Sequence of the H1.01 locus and its processed RNAs. The genomic sequence and numbering are taken from Harvey et al. (19821 and Coles and Wells (19851. The chicken histone H1 consensus sequences for various promoter e lements {Coles et al. 19871 and the HI.01 init iation and stop codons and 3'-processing e lements {Coles and Wells 1985), as wel l as the 3'-processing e lements of the convergently transcribed H2A gene (Harvey et al. 19821, are underlined. The H1.01 transcriptional start site is marked wi th a bent arrow. The poly(A) + c D N A sequen.ce begins at nucleot ide 708 {*) and is identical to the genomic sequence, except at GC 1669-1670 , where it is CG, and at T 1718, where the c D N A contains a G. The first A of the c D N A poly(A1 tail is at posit ion 2173 {*). Direct repeats f lanking the pseudogene-l ike e lement are labeled DR 1 and DR 2 and are boxed, as are the init iat ion and stop codons, poly(A) addition signal, and genomic poly(A) tract of the pseudogene- l ike e lement. The pseudogene-l ike element's open reading frame is translated. The sites of histone-specif ic pre-mRNA cleavage are indicated wi th vertical arrows. (B) Line diagram of the H1.01 locus, comparing the sequence content of the poly(AI- m R N A and the poly(A) + cDNA, and the predicted extent of the 2-kb polyIA I + m R N A . The poly(A) tails of the c D N A and m R N A are filled and are drawn to the length of the c D N A poly(AI tail.





Kirsh et al.

transcript is polyadenylated (Fig. 5). We screened the myotube cDNA library with H1.10.907 and identified a cDNA with 935 bases of HI .10 sequence 5' to the poly(A) tail, including an internal s t em- loop and purine-rich box (Fig. 6). Polyadenylation of the transcript involves the pseudogene-like element 's AATAAA hexamer, 160 nucleotides 3' to the histone-specific cleavage site, indicating that this RNA and the H1.01 2-kb RNA may arise through a similar mechan ism of alternative 3 '-end formation.

Discussion

In this paper we describe histone H1 mRNAs that are polyadenylated and retain internal 3' processing sequences. The H1.01 and HI.10 genes are the first endog- enous histone genes found to use two modes of 3' processing in somatic tissues. Other poly(A) + histone transcripts wi th internal processing sites have been described previously, some of them arising from experi- menta l rather than natural situations. For example, A1- terman et al. (1985) reported a minor poly(A) + transcript synthesized from a chimeric mouse H3 gene transfected into mouse fibroblasts. The polyadenylation of these chimeric transcripts involves a cryptic AATAAA hexamer in flanking sequences downstream of the inte- grated gene. These poly(A) ÷ transcripts are stable and cytoplasmic. Their existence is at least partly dependent on the high copy number of the introduced construct, which probably effects a t i tration of histone-specific cleavage factors. Similar RNAs are expressed by an (x- globin/H4 hybrid gene in which the globin polyadenylation signal is dominant in dividing cells when placed downstream of the histone-specific cleavage sequences (Whitelaw et al. 1986).

Recently we have described natural ly occurring

/ y-

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Figure 5. Northern analysis of HI.10 gene transcripts. Fifteen micrograms each of spleen and skeletal muscle RNA were loaded onto the first two lanes of the gel. The filter was probed with the H1.10 gene-specific oligonucleotide HI. 10.907, which is complementary to sequence in the 3'-nontranslated region, 5' to the stem-loop. Eighteen micrograms of poly(A)- RNA and 3.5 ~g poly(A) + RNA from cultured myotubes was loaded onto the last two lanes of the gel. The filter was probed with the HI.10 gene-specific oligonucleotide H1.10.5', which is complementary to sequence in the 5' leader.

2176 GENES & D E V E L O P M E N T

AAGCTTCAAGGTCTTCCCTTCACCCCCTGAAGAAAGTGGGGTGATTTCGAGCCCGGCATT 60

A-RICH TTCCAAAAAACACGAATTTATAACTCCGAAGAAACACAGACTCGGAGGACGGAAAGCTCT 120

G-RICH CCAAT TATA TCCTGGCTAACAGTTAGGCGGGCTCTGCAAAGCACCAA~CACAGATCACCGCTTCGCTAT 180

AAATTCAGGCATCGGGGTTACTGTAGCCCAATTACTTTCTTTTGATTAGGAAGAAGTCTC 240 TGCCGCGATGTCGGAGACCGCTCCCGCCGCCGCTCCCGCTGTCGCGNCCCCCGCCGCCAA 300 GGCCGCCGCCAAGAAGCCGAAGAAGGCGGCGGGCGGCGCCAAAGCCCGCAAGCCCGCGGG 360 CCCCAGCGTCACCGAGCTGATCACCAAGGCCGTGTCCGCCTCCAAGGAGCGCAAGGGGCT 420 CTCCCTCGCCGCGCTCAAGAAGGCGCTGGCCGCCGGCGGCTACGACGTGGAGAAGAACAA 480 CAGCCGCATCAAGCTGGGGCTCAAGAGCCTCGTCAGCAAGGGCACCCTGGTGCAGACCAA 540 GGGCACCGGCGCCTCGCGCTCGTTCCGTCTCAGCAAGAAGCCGGGTGAGGTGAAGGAGAA 600 G G C T C C C A G G A A G A G A A C G C C C G C G G C C A A G C C C A A G A A G C C G G C G G C G A A G A A G C C T G C 6 6 0

C A G C G C C G C C ~ G C C C ~ G ~ G G C O G C G G C G G C O a a G ~ G A G C C C ~ G ~ G C C ~ 72O GA.AGCCGGCGGCTGCCGCCACCMGMGGCGGCCMGAGCCCCMGMGGCTACCMGGC 780 TGCCAAGCCCAAAAAGGCGGCGACTGCCAAAAGCCCGGCCAAGGCAAAGGCGGTGAAGCC 840 CAAAGCTGCCAAGCGCAAGGCGGCCAAACCCAAGGCAGCCAAGGCGAAGAAGGCGGCGGC 900

HI DYAD ELEMENT CAAGAAGTGAAGTATTGAAGTGGAAATACTTAAACCCAACGGCTCTTTTAAGAGCCACCC 960

PURINE BO ACCACTGTCCGAAAAAGA~ACACT~'-~TGAGCTCC~CTGCAGCGAACG 1020 - M P A A N V

TCACGATTCGCCATTATTTGCTCGCGATCGGAGTATTTTGCTCGCCTGCAAAAAGCGCGG 1080 T I R H Y L L A I G V F C S P A K S A G

GGCTC~CTGCCGGACGTGGTTAAAATA~'~~TTTGCGCACTCT 1140 L

GGG~T~*~T~T~4--~CT~'G~ACGG~d&4~ 1200 GGTGCGTGTG 1210

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Figure 6. (A) Sequence of the HI.10 gene and its processed RNAs. The genomic sequence and numbering are taken from Coles et al. (1987). Regulatory elements are labeled as in Fig. 4. The cDNA sequence begins at C 238 (St) and is identical to the genomic sequence, except at 734-735 where the cDNA sequence is CG. We assume that this is a cloning artifact. N 287 in the genomic sequence is a G in our cDNA. The first A of the cDNA poly(A) tail is at position 1145 (St). The first direct repeat flanking the pseudogene-like element overlaps the HI.10 purine-rich box by 3 bases. (B) Line diagram of the H1.10 locus, comparing the sequence content of the poly(A)- mRNA and the poly(A) +cDNA, and the predicted extent of the poly(A) + RNA. The poly(A) tails of the eDNA and mRNA are filled and are drawn to the length of the eDNA poly(A) tail.

poly(A) + H2a, H2b, and H3 mRNAs that retain internal 3'-processing signals in rooster spermatids (Challoner et al. 1989). In this instance, AATAAA-like sequences are included in the purine-rich boxes of the H2b genes, and sequences related to the second polyadenylation signal consensus element YGTGTTYY (McLauchlan et al. 1983) are found a short distance downstream. Tran- scripts from the same genes in somatic cells are poly(A)-, contain the 3 ' - terminal s t em- loop , and are regulated during the cell cycle like other replication-dependent histone genes. Expression of the polyadenylated H2b mRNAs is restricted to the germ line. In contrast, the poly(A) + H1.01 and HI .10 transcripts are found in quiescent and differentiated cells but not in spermatids (data not shown). This difference in tissue distribution suggests that different mechanisms of alternative 3'-end




formation operate either on the chicken H2b and HI genes or in quiescent versus germ line cells, or both.

The presence of abundant, stable H1.01 and HI.10 polyadenylated mRNAs in tissues that are enriched in c fraction H1 subtypes suggests that alternative 3' processing may contribute to the expression of these genes in the absence of DNA replication. The coexistence of alternative RNA-processing products at different levels in a variety of cell types suggests that the two reactions, cleavage at the s t em- loop and cleavage and polyadenylation, may be in competition. Certain features of the H1.01 and HI.10 sequences are l ikely to be involved in determining the ratio of poly(A)- to poly(A) + mRNA synthesized by these genes. Nei ther of the H 1 polyadenylation signals includes a YGTGTTYY sequence, which has been shown to increase the efficiency of pre-mRNA cleavage and polyadenylation (McLauchlan et al. 1983). The globin/H4 hybrid gene noted above, in which polyadenylation was the dominant mode of processing in dividing cells, included this sequence in its polyadenylation signal. The absence of this e lement from H1.01 and HI.10 may be important in allowing U7 snRNP-mediated 3' processing to be the predominant mode of 3' processing in dividing cells. In this view, polyadenylation may be the default mode of processing in nondividing cells, resulting from the reduction in activity of one or more components of the U7 snRNP-associated reaction. The heat-labile activity (Luscher and Schum- perli 1987; Stauber and Schumperl i 1988) that l imits cleavage in Gl-arrested cells is a candidate for such a l imit ing component. The proximity of the poly(A) addition signal used by the H1.01 gene to the 3' end of the convergently transcribed H2A gene (Fig. 4A) also raises the possibil i ty that transcription from the H1.01 promoter through the AATAAA element may be inhibited during S phase by increased opposing transcription from the H2A promoter. This may favor the processing of precursors to form poly(A)- mRNAs in dividing cells.

As noted by Coles et al. (1987), the H1.01 purine-rich box differs from the chicken H1 consensus by the inser- tion of a G in the sequence predicted to base-pair with the U7 snRNA (Mowry and Steitz 1987a). Partial desta- bil ization of binding of the U7 snRNP to the H1.01 purine-rich box may favor the expression of polyadenylated mRNAs from this gene by reducing the efficiency of histone-specific cleavage. The H I.10 gene's purine-rich box matches the consensus, and this may explain the l imited tissue distribution and low level of expression of polyadenylated versus nonpolyadenylated HI.10 transcripts, relative to the H1.01 gene.

The polyadenylation signals used by the H1.01 and HI.10 genes are unusual in their association wi th sequences resembling processed pseudogenes. Such an or- igin would explain the lack of YGTGTTYY elements, as these are not present in mature polyadenylated transcripts and would thus not be available to be copied from a putative RNA precursor into processed pseudogene DNA. To our knowledge, this is the first description of a pseudogene-like sequence supplying a regulated, cis- acting e lement to an adjacent gene. The translational

Alternative RNA processing in avian histone HI genes

start sites of both the pseudogene-like sequences lack the purine at position - 3 and the G at + 4, which are thought to be necessary for efficient ini t iat ion (Kozak 1989), and the codon usage of both open reading frames is not s imilar to that of any known organism. Transla- t ion of these reading frames in the polyadenylated transcripts thus seems unlikely. The H1.01 e lement ' s predicted amino acid sequence does include a region (en- coded by nucleotides 1919-1946 and containing the core consensus G-X-G-X-X-G) s imilar to the amino- proximal portion of an ATP-binding motif common to protein serine-threonine and tyrosine kinases (Hanks et al. 1988), but it lacks the essential conserved lysine found approximately 15 amino acids carboxy-terminal to the third glycine. Comparison of the predicted amino acid sequences of both of the pseudogene-like elements ' open reading frames wi th sequences in the Protein Iden- tification Resource and EMBL databases has revealed no extensive similari t ies to known proteins.

E x p e r i m e n t a l p r o c e d u r e s

Muscle culture

Primary cultures of cells from l 1-day chick embryo breast muscle were prepared according to the protocol of Bischoff and Holtzer (1967) and grown in Dulbecco's modified Eagle medium (DMEM) with 10% horse serum and 10% embryo extract. Fusion of myoblasts into myotubes was evident at 2 days postplating. RNA was prepared from cultures at 4 days. Alterna- tively, at 3 days postplating, to eliminate remaining mitotically active cells, some cultures were treated with fresh medium containing 10p.M cytosine arabinoside for 2 successive days and were then returned to drug-free medium for 1 day before RNA preparation. Control cultures were given the same regimen but without the drug.

Chickens

White leghorn roosters, 6 months to 1 year old, were the source of adult tissues for RNA analysis.

RNA preparation

Total RNA from cultured cells, tissues, and fractionated polysomes was prepared by the guanidinium isothiocyanate proce- dure (Maniatis et al. 1982). Selection of poly(A) + RNA on oligo(dT)-cellulose (Collaborative Research) was performed according to Kingston (1987).

mRNA half-life measurements during the cell cycle

Primary cultures of chick embryo fibroblasts {Linial and Mason 1973) were grown to confluence in medium with 6% serum, maintained in medium with 0.5% serum for 12 hr, and incubated in fresh medium containing 6% serum. Following this serum stimulation, -80% of these cells synchronously traverse the cell cycle (Thompson et al. 1985). To measure the stability of histone transcripts during the cell cycle, RNA was prepared from cells that were treated for 0, 45, 90, and 135 rain with 5 p.g/ml actinomycin D after 0 and 10 hr of serum stimulation, when the cells were in Go and early S phase, respectively (Thompson et al. 1985). Ten micrograms of each RNA sample was separated on a 1.2% agarose/formaldehyde gel for Northern analysis.





Kirsh et al.

Polyribosome analysis

A modified version of the protocols of Heywood et al. (1968) and Brown et al. (1974) was used to prepare polysomes from the breast muscles of 14-day chick embryos. Buffers used were buffer G [250 mM KC1, 10 mM MgClz, 20 mM vanadyl ribonu- cleosides (Sigma), 10 mM Tris-HC1 at pH 7.4]; buffer F (25 mM KCI, 5 mM MgC12, 50 mM Tris-HC1 at pH 7.8); buffer R (50 mM KC1, 25 mM MgC12, 50 mM Tris-HC1 at pH 7.4); and buffer P (25 mM KCI, 25 mM MgC12, 50 mM Tris-HC1 at pH 7.4). All steps were performed at 0-4°C, unless otherwise noted. Breast muscle from six embryos was minced in 3 volumes buffer G, homogenized with two strokes of a B pestle in a Dounce ho- mogenizer, and spun for 10 rain at 10,000g. The supernatant was overlaid on a 3-ml cushion of 1.5 M sucrose in buffer F in a 14-ml tube. The tube was filled with buffer G and then spun for 19.5 hr at 35,000 rpm in a Beckman SW 41Ti rotor. The pelleted polysomes were resuspended in buffer R, frozen in dry ice/eth- anol, and stored at - 70°C for less than 1 week before fraction- ation. Thawed polysomes were made 1 M in KC1 and 8.3 mM in EDTA. For puromycin treatment, samples were made 2 mM in puromycin (Sigma). All samples were incubated on ice for 15 min, followed by 10 min at 37°C. Five A26o units of polysomes were loaded per 10-50% continuous sucrose gradient in buffer P and spun for 1 hr at 47,000 rpm in a Beckman SW 55Ti rotor. Fractions were collected directly into guanidinium isothiocyanate buffer at room temperature from the tops of the gra- dients using an Isco model 185 fraction collector with a flow cell. Absorbance was recorded with a UA5 absorbance monitor at 254 nm. RNA was prepared from the fractions as described above.

Myotube cDNA library

The eDNA library derived from poly(A) + RNA from cultured embryonal myotubes and packaged in hgtl0 was a gift from Dr. Bruce Paterson.

Filter hybridizations, probes, and DNA sequencing

Northern and plaque hybridizations on nitrocellulose {Schleicher & Schuell) were performed as described previously (Challoner et al. 1989), with one modification: Filters hybridized to oligonucleotide probes were given an additional 1-min wash at the hybridization temperature. Blotting of polyribosomal fraction RNA onto nitrocellulose was performed with a Slot Blotter (Schleicher & Schuell). Oligonucleotides were synthesized on an Applied Biosystems model 380B DNA synthe- sizer. Subcloning of phage genomic and cDNA clones into the vector pGEM2 {Promega Biotec), 32P-labeling of oligonucleotides with T4 polynucleotide kinase, and nick-translation of plasmid inserts isolated by agarose gel electrophoresis were performed as described (Maniatis et al. 1982). The following DNAs were used as hybridization probes:

1. pCH42 insert: The 2066-base EcoRI-SmaI fragment from hGhSe (Sugarman et al. 1983), which includes the H1.01 gene, 500 bp of 5'-flanking sequence, and 3' sequence extending to base number 2066 in Figure 4, corresponding to bases 1-2066 in the sequence of Coles and Wells (1985}.

2. pCH42.8 hTsert: The AluI-AccI fragment extending from position 1399 to 1544 in Figure 4, entirely 3' to the H1.01 histone-specific cleavage site.

3. Hl.O1.3': H1.01-specific oligonucleotide probe complementary to nucleotides 1324-1343 in the 3'-nontranslated region (see Fig. 4) 5'-TAGAGCAGGACTCTTCTGGG-3'.

4. H1.10.907: Hl.10-specific oligonucleotide probe complementary to the last nucleotide of the Hl.10-coding region and 19 nucleotides of the noncoding region [position

2178 GENES & DEVELOPMENT

907-926 in Coles et al. (1987) and Figure 6; 5'- TTTCCACTTCAATACTTCAC-3'].

5. H1.10.5': H 1.10-specific oligonucleotide probe complementary to the 5'-leader sequence, comprising nucleotides 227-246 in Coles et al. (1987) and Figure 6 (5'-GCGGCA- GAGACTTCTTCCTA-3').

A Sequenase kit (United States Biochemical) was used for di- deoxy sequencing of cDNA clones in pGEM2.

A c k n o w l e d g m e n t s

We thank B. Paterson for the generous gift of the cDNA library, S. Tapscott for advice on muscle culture, P. Finerty for plasmid preps, and A. Lassar, M. Linial, S. Moss, H. Sire, and H. Wein- traub for comments on the manuscript. This work was funded by National Science Foundation grant DCB8802490 and U.S. Public Health Service Program Project grant CA28151 to M.G. P.B.C. is a special fellow of the Leukemia Society of America. A.L.K. was supported by National Institutes of Health Training grant T32-CA09437.

Note added in proof

While this paper was in press, G. Cheng, A. Nandi, S. Clerk, and A.I. Skoultchi (1986) reported that the mouse Hl-var. 1 gene (which lacks any pseudogene-like element) is expressed as both poly{A)- and poly(A) + mRNAs with properties similar to those described in this work. Together these results suggest that alternative 3' processing of mRNA may be a common regulatory feature of vertebrate H1 gene expression.

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10.1101/gad.3.12b.2172Access the most recent version at doi: 3:1989, Genes Dev.

A L Kirsh, M Groudine and P B Challoner of RNA 3' processing in two avian histone H1 genes.Polyadenylation and U7 snRNP-mediated cleavage: alternative modes

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