snRNA genes: a model system to study fundamental mechanisms of transcription

Nouria Hernandez

Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

11724.

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Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on June 4, 2001 as Manuscript R100032200 by guest on M

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The human small nuclear RNA (snRNA) genes, which encode snRNAs that are involved in RNA

processing reactions such as mRNA splicing, serve as prototypes for a family of genes whose

promoters are characterized by the presence of a proximal sequence element (PSE) and a distal

sequence element (DSE). From a transcription point of view, this family of genes is highly

interesting because all its members have very similar promoters, even though some of them are

transcribed by RNA polymerase (pol) II and others by pol III. As a result, the snRNA genes have

served as a model system to explore how RNA polymerase specificity is determined, and, in

general, to compare the pol II and III transcription machineries. This has led to the concept that

the pol II and III transcription machineries use common factors, the best known of which is the

TATA box binding protein TBP. In addition, the relative simplicity of these promoters has also

made them an attractive system to study how transcriptional activators perform their function.

The structure of snRNA promoters.

Figure 1 shows the structures of snRNA promoters from Homo sapiens (Hs), Arabidopsis

thaliana (At), and Drosophila melanogaster (Dm), and serves to illustrate the remarkable fact that

although snRNA promoters have diverged during evolution, the close similarity between those

recognized by pol II and those recognized by pol III has been conserved. In fact, in each of the

examples in Figure 1, RNA polymerase specificity can be changed by altering a single

parameter, indicated in red in the figure.

In the human genes, the U1 and U2 snRNA promoters serve as the prototypic pol II snRNA

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promoters, and the U6 snRNA promoter serves as the prototypic pol III snRNA promoter (see (1)

for a review). The human pol II snRNA core promoters contain only one essential element, the

proximal sequence element (PSE), whereas the pol III snRNA core promoters consist of two

elements, the PSE and a TATA box located at a fixed distance downstream. The distal sequence

element (DSE) serves to enhance transcription from the core promoter. Both the DSE and the

PSE can be interchanged between pol II and III snRNA promoters with no effect on RNA

polymerase specificity, which is determined by the presence or absence of the TATA box. The

A. thaliana pol II and III snRNA promoters contain an upstream sequence element (USE) and a

TATA box, which are both interchangeable between the pol II and III snRNA promoters. RNA

polymerase specificity is determined in this case by the exact spacing between the USE and the

TATA box, which is 33 to 34 base pairs (bp) and 23 to 24 bp in the pol II and III snRNA

promoters, respectively (2).

The D. melanogaster pol II snRNA promoters contain two elements referred to as the PSEA and

the PSEB spaced by eight bp, and the pol III snRNA promoters contain a PSEA and a TATA box

spaced by 12 bp. The PSEA is quite conserved in various pol II and III snRNA promoters but

positions 19 and 20 of the 21 bp elements are always g/aG in the pol II, and TC in the pol III,

snRNA promoters. RNA polymerase specificity is determined by the precise sequence of the

PSEA element, with the base pairs at positions 19 and 20 playing a major role ((3), and

references therein).

A number of snRNA promoters have been characterized in various sea urchins. Like in other

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species, the pol II and III snRNA promoters are closely related in structure. They all have a PSE

and some have a TATA box, but the presence of the TATA box does not correlate with RNA

polymerase specificity. The PSEs in different snRNA promoters show little sequence identity

and yet can be exchanged with no effect on polymerase specificity. The determinants of RNA

polymerase specificity are not known ((4), and references therein). In Saccharomyces cerevisiae,

only the pol III U6 snRNA promoter has been studied. It consists of a TATA box located

upstream of the transcription start site, and A and B boxes typical of gene-internal tRNA

promoters. The A box is located, as in tRNA genes, within the RNA coding region, but the B

box is located at an anomalous position 3’ of the gene (5-7).

The PSE binding factors.

The snRNA promoters in vertebrates, A. thaliana, D. melanogaster, and sea urchins all contain an

element, variously called the PSE, PSEA, or USE, centered 50 to 70 bp upstream of the

transcription start site. The factor binding to this element has been best characterized in the

human system, and is variously known as PBP, PTF, or SNAPc. It is a complex containing five

types of subunits, SNAP190, SNAP50 (PTFβ), SNAP45 (PTFδ), SNAP43 (PTFγ), and SNAP19

(see (8) and references within). SNAP190 forms the backbone of the complex, with SNAP19

and SNAP45 associating toward the N- and C-terminus, respectively, of the molecule. SNAP43

can associate with the same region of SNAP190 as SNAP19, and SNAP50 joins the complex by

associating with SNAP43 (see Figure 3 below for an illustration SNAPc).

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The ability to assemble recombinant SNAPc and, thus mutant forms of SNAPc has allowed a

study of the role of SNAPc subunits for binding to DNA, and for basal and activated

transcription by RNA polymerases II and III. The smallest subassembly of SNAPc subunits

tested that binds to the PSE with the same specificity as the complete complex consists of

SNAP190 aa 84-505, SNAP43 aa 1-268, and SNAP50 (9). This observation is consistent with

UV crosslinking experiments that suggest that within SNAPc, both SNAP190 and SNAP50 are

in close contact with the DNA (see (8) and references therein). The specific binding of SNAPc

to the PSE is mediated in part by an unusual Myb domain extending from aa 263 to 503 within

SNAP190 and containing a half repeat followed by four repeats (8,10).

In the human system, the very same SNAPc is involved in transcription by pol II and pol III (11).

The polypeptide composition of PSE-binding factors from other species is unknown, but there

are indications that at least in some cases, the same PSE binding factor is also recruited to both

pol II and III snRNA promoters. Thus, both in sea urchins and D. melanogaster, similar

complexes bind to the PSEs of pol II and III snRNA promoters as judged from electrophoretic

mobility shift assays (EMSAs) (3,4), and in the latter case, site-specific protein-DNA photo

cross-linking experiments reveal the same set of polypeptides in close proximity to the DNA in

both cases. Interestingly, however, the precise crosslinking patterns of these polypeptides to the

U1 and U6 PSEAs are significantly different. Thus, in D. melanogaster, RNA polymerase

specificity may ultimately be determined by different conformations of the same factor, which

are dictated by the exact PSEA sequence (12).

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Factors besides SNAPc required for pol II transcription of snRNA genes.

Transcription from TATA-box containing mRNA promoters can be reconstituted with a

combination of recombinant and well-defined factors, as shown in Figure 2A. In vitro, these

factors can be added sequentially to the promoter to form a functional transcription initiation

complex, and each step can be monitored by EMSA. TBP or the TBP-containing complex

TFIID binds first to the TATA box, followed by TFIIB, a TFIIF/Pol II complex, TFIIE, and

TFIIH. TFIIA can join the initiation complex at any stage of assembly, and its main role for

mRNA core promoter function appears to be counteracting repressors that associate with TBP

and prevent its binding to DNA (13).

In the case of the pol II snRNA promoters, the transcription initiation complex has not yet been

assembled in a stepwise fashion in vitro, but many of its components have been identified

functionally by depletion of transcription extracts with specific antibodies and reconstitution of

transcription with recombinant factors. Thus, many of the players are known but their mode of

assembly on snRNA promoters is not, and thus their location in Figure 2A is arbitrary.

Depletion and reconstitution experiments indicate that recombinant TBP (but not the TBP-

containing complexes TFIID or TFIIIB), TFIIB, TFIIA, TFIIF, and TFIIE are required ((14), and

references therein). TFIIA appears to perform a more direct function in snRNA transcription

complex assembly than just counteracting TBP-associated repressors. The role, if any, of TFIIH

in pol II transcription of snRNA genes is not clear. Depletion and reconstitution experiments

suggest that U1 transcription either does not require TFIIH, or requires much lower levels than

transcription from a mRNA promoter. If TFIIH is indeed not required, this raises the interesting

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question of how open complex formation is achieved at snRNA promoters. A combination of all

the general transcription factors and SNAPc does not initiate transcription from the U1 promoter,

suggesting that additional as yet unidentified factors are required (14).

Factors besides SNAPc required for pol III transcription of snRNA genes.

The key player for recruitment of pol II to a promoter is the factor TFIIB, because it contacts the

RNA polymerase directly. In the case of pol III, this role is played mainly by the multisubunit

factor TFIIIB. TFIIIB was completely defined first in S. cerevisiae and consists of three

subunits, TBP, a tightly associated subunit referred to as the TFIIB-related factor BRF1

(PCF4/TDS4) (see (15) for references), and a more loosely associated polypeptide called B"

(TFIIIB90/TFC5/TFC7) (16,17). This TFIIIB complex is involved in transcription from all types

of yeast pol III promoters tested including the gene-internal tRNA-type promoters and the U6

promoter, which, as described above, contains a TATA box and A and B boxes (18).

TBP was shown to be required for pol III transcription of vertebrate snRNA genes before TBP

was known to be a subunit of TFIIIB (see (15) for a review). Ironically, however, the

composition of mammalian TFIIIB and the role of TFIIIB polypeptides other than TBP in

snRNA gene transcription have been determined only recently. A human homologue of yeast B"

was recently cloned (19). The protein shows strong similarity to the yeast protein within and

around a 59 aa domain called the SANT domain, which is essential for transcription in yeast.

Depletion of human B" (hB") from transcription extracts debilitates transcription from the U6

promoter and a tRNA-type promoter, and transcription can be restored in both cases by addition

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of recombinant hB" (19). hB" is, therefore, shown as part of the initiation complex assembled on

both U6 and tRNA-type promoters in Figure 2B.

The first human homologue of yeast BRF cloned was called TFIIIB90 (20) or human BRF

(hBRF) (21). Like its yeast counterpart, and like its cousin TFIIB, the protein has a zinc binding

domain at its N-terminus followed by a core domain consisting of two degenerate repeats. The

C-terminal half of the protein is poorly conserved with the yeast protein, and has no counterpart

in TFIIB. Depletion and reconstitution experiments have shown that human BRF is required for

transcription from tRNA-type promoters, but not for transcription from the U6 snRNA promoter

(21). Remarkably, as shown in Figure 2B, the U6 snRNA promoter uses another homologue of

yeast BRF, human BRFU (19), also called TFIIIB50 (22). hBRFU represents another member of

the TFIIB-related family of proteins and has conserved zinc and core domains, and a divergent

C-terminal domain.

Human BRFU was cloned both through database searching of proteins similar to hBRF (19) and

through purification of a complex consisting of BRFU and four tightly associated polypeptides

(22). The role of the BFRU-associated polypeptides is presently not clear. In one series of

experiments, U6 transcription could be restored in a BRFU-depleted extract by addition of

recombinant BRFU expressed in E. coli (19), and a combination of partially purified pol III and

recombinant SNAPc, TBP, hB", and hBRFU could direct efficient U6 transcription (23). In

another series of experiments, depletion of BRFU (TFIIIB50) debilitated transcription from a

snRNA-type promoter, but transcription could not be reconstituted by addition of recombinant

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BRFU. Instead, transcription could only be reconstituted by addition of a BRFU-containing

complex immuno-purified from cells expressing tagged BRFU (22). Thus, it is not clear

whether the BRFU-associated polypeptides in the BRFU-containing complex are essential for

U6 transcription.

BRF2, another factor encoded by an alternatively spliced BRF pre-mRNA, may also be required

for U6 transcription (24). BRF2 lacks the zinc finger domain and the first repeat that are present

in BRF and conserved in the other proteins of the TFIIB family, as well as the C-terminal

domain present in BRF. Depletion of extracts with antibodies recognizing all BRF variants

debilitated U6 transcription, and transcription could be specifically reconstituted by addition of

material immuno-purified from cells expressing tagged BRF2 (24). These results raise the

possibility that the U6 transcription complex contains two proteins related to BRF, BRFU and

BRF2.

The discovery of BRFU is an important step toward a complete understanding of how RNA

polymerase specificity is determined at the human snRNA promoters. Indeed, in mRNA

promoters, TFIIB associates with TBP bound to the TATA box and, in a manner absolutely

dependent on the zinc ribbon domain, with the pol II/TFIIF complex (13). Thus, at least for

mRNA promoters, TFIIB can be viewed as the key factor that bridges DNA-associated TBP or

TFIID with the polymerase, and it seems likely that TFIIB performs the same role in the pol II

snRNA promoters. Like TFIIB, BRF also contacts directly the RNA polymerase, in this case pol

III. The precise BRF domain required for this function is not known but it is not the zinc ribbon

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domain, because deletion or mutation of the zinc ribbon does not affect RNA polymerase

recruitment but instead affects open complex formation (see (19) for references). It seems likely

that in the human U6 promoter, the recruitment of pol III is accomplished by BRFU, either

through the zinc domain as for TFIIB and pol II, or through another part of the protein as for

BRF and pol III at tRNA-type promoters. Thus, the determination of RNA polymerase

specificity may ultimately depend on whether TFIIB or BRFU is recruited to the promoter.

Activation of snRNA gene transcription.

The human snRNA promoters are activated by a DSE. The DSE is composed of various protein

binding sites, but one of them is almost invariably the octamer sequence ATGCAAAT. In

addition, it has become clear that the DSEs of many snRNA genes contain an element referred to

as the SPH element. The SPH (for “Sph1 postoctamer homology”) element was first identified

in the chicken snRNA gene enhancer ((25,26), and references therein), and in the enhancer of the

selenocysteine tRNA gene, whose promoter contains a PSE and TATA box (27,28). It then

became clear that a functionally important element located immediately upstream of the octamer

sequence in the human U6 snRNA promoter called the NONOCT element (29) corresponds, in

fact, to an SPH element (30), and that SPH elements are present in the enhancers of many

snRNA promoters (31). In the human U6 snRNA promoter, both the octamer and SPH elements

stimulate the formation of preinitiation complexes (32).

The SPH motif recruits in vitro a transcription factor called Staf or SPH binding factor (SBF),

which was cloned first from Xenopus (31,33), and then from mouse (34) and humans (35,36).

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Xenopus Staf is a zinc finger protein containing seven zinc fingers of the C2-H2 type, different

sets of which can be used to bind to different DNA targets. In particular, zinc finger 1 is required

for binding to the selenocysteine tRNA enhancer but not to the U6 enhancer, where introduction

of a zinc finger 1 binding site interferes with binding of Oct-1, the protein recruited to the

adjacent downstream octamer sequence ((37) and references therein). Xenopus Staf contains two

separable activation domains capable of selectively stimulating transcription from snRNA-type

and mRNA-type promoters (38). The human proteins ZNF143 and, to a lesser extent, ZNF76,

are similar to Xenopus Staf, share similar DNA binding specificities, and can activate pol II and

III snRNA gene transcription (35,36).

The octamer sequence recruits the transcription activator Oct-1, as suggested by i) the broad

expression of Oct-1, which parallels the broad expression of snRNA genes; ii) the presence of

snRNA-specific transcription activation domains within Oct-1; and iii) the localization of Oct-1

to snRNA promoter sequences in vivo by chromatin immunoprecipitation experiments ((39), and

references therein; (40)). Oct-1 activates snRNA gene transcription not only through its

activation domains, but also through its POU domain, a bipartite DNA binding domain

consisting of two helix-turn-helix-containing DNA-binding structures: an amino-terminal

POU-specific (POUS) domain and a carboxy-terminal POU-homeo (POUH) domain, joined by

a flexible linker (41). As described further below, this results from the ability of the Oct-1 POU

domain to bind cooperatively with SNAPc and thus recruit SNAPc to the PSE.

Assembly of a stable snRNA transcription initiation complex.

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The characterization of many of the factors that bind to snRNA promoters has allowed a study of

how these factors interact with each other to form a stable transcription initiation complex. Our

current understanding of this process is summarized in Figure 3. Both TBP and SNAPc have

built in mechanisms that prevent their efficient binding to DNA on their own. In the case of

TBP, this “damper” of DNA binding resides in the N-terminus of the protein, because deletion

of this segment greatly increases the ability of the truncated protein to bind to TATA boxes (42).

Perhaps the N-terminus of TBP masks the DNA binding domain of the protein, as illustrated in

Figure 3A, although other scenarios are equally possible. In the case of SNAPc, the damper of

DNA binding resides somewhere within the C-terminal two thirds of SNAP190 and/or SNAP45,

because a mini-SNAPc lacking these sequences binds much more efficiently to DNA than

complete SNAPc (10). Both TBP and SNAPc dissociate slowly from the DNA and bind with

relatively low sequence specificity, so these built-in dampers may serve to ensure that these

factors not bind to inappropriate sites in the genome.

Figure 3B illustrates SNAPc, TBP, and the Oct-1 POU domain bound to DNA. The Oct-1 POU

domain and SNAPc bind cooperatively to their respective DNA binding sites, and so do SNAPc

and TBP (see (8) and references within). Very strikingly, the same regions of SNAPc and TBP

that serve as dampers of DNA binding are required for cooperative binding. Thus, the N-

terminal domain of TBP is absolutely required for cooperative binding with SNAPc, perhaps

because as illustrated in Figure 3B, it is engaged in a protein-protein interaction with SNAPc

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(42). Similarly, the C-terminal domain of SNAP190 is required for cooperative binding with

Oct-1, and in this case it is clear that cooperative binding is dependent on a direct protein-

protein contact between the two proteins, which involves a glutamic acid at position 7 within the

POUS domain (blue triangle in Figure 3B) and a lysine at position 900 within SNAP190 (Oct-1

interacting region “OIR” in Figure 3B) (43). Thus, cooperative binding of these factors probably

involves conformational changes that convert the dampers of DNA binding into handles that

contact and stabilize the factor binding to a neighboring site. This intricate mode of DNA

binding ensures, in effect, that factors bind to sites located in promoter sequences rather than to

inappropriate isolated sites.

The cooperative binding of the Oct-1 POU domain and SNAPc was originally characterized on

probes containing closely spaced octamer and PSE. In the natural snRNA promoters, however,

the octamer sequence and the PSE are separated by about 150 bp, and this distance prevents

cooperative binding of Oct-1 and SNAPc on naked DNA probes. However, mapping of DNase

I and micrococcal nuclease cleavage sites in chromatin suggests the presence of a positioned

nucleosome between the octamer sequence and the PSE in both the U1 and U6 snRNA

promoters (40,44). Indeed, in vitro chromatin assembly results in the positioning of a

nucleosome at the same location (40,45). Importantly, chromatin assembly allows the Oct-1

POU domain to activate transcription from the natural U6 promoter. It also allows cooperative

binding of the Oct-1 POU domain and SNAPc, and this cooperative binding is dependent on the

same direct protein-protein contact as cooperative binding to closely spaced sites on naked DNA

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(40). These results suggest that the role of the positioned nucleosome is to bring into close

proximity the octamer sequence and the PSE such that SNAPc and the Oct-1 POU domain can

contact, and recruit, each other to the DNA, as illustrated in Figure 3B. Thus, this is a case

where a nucleosome does not repress transcription but instead is a functional component of the

transcription activation process.

ACKNOWLEDGMENTS

I thank Winship Herr for comments on the manuscript. Our work on snRNA gene transcription

is funded in part by NIH grant GM38810. N.H. is supported by the Howard Hughes Medical

Institute.

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FIGURE LEGENDS

Figure 1. Structure of the Homo sapiens (Hs), Arabidopsis thaliana (At), and Drosophila

melanogaster (Dm) snRNA promoters. See description in the text.

Figure 2. Composition of pol II and III transcription initiation complexes. A). pol II initiation

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complexes assembled on a TATA-box containing mRNA core promoter and on the human U1

snRNA core promoter. B). pol III initiation complexes assembled on the human U6 snRNA

promoter and a tRNA-type promoter. The placement of hBRF, hBRFU, and hB" is arbitrary. A

stippled line separates TBP and hBRF to indicate that these factors are tightly associated with

each other in solution. In contrast, there is no evidence that TBP and hBRFU are associated with

each other in solution.

Figure 3. Assembly of a stable snRNA transcription initiation complex. A. TBP and SNAPc

contain built-in dampers of DNA binding that downregulate their binding to DNA. B.

Assembly of the U6 initiation complex. See text for description.

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DSE PSE

TATADSE PSE

33-34

23-24

USE TATA

USE TATA

PSEA PSEB

ga G

PSEA TATA

TC

pol II

pol III

pol II

pol III

pol II

pol III

Hs

At

Dm

CORE PROMOTER REGION

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IIA

IIA

IIE

IIFIIB

Pol 2TBP

TATA

SNAP c

PSEU1

mRNA

IIH

?TBP

IIE

IIFIIB

Pol 2

IIH

TFIIIC

A B

TFIIIB

TBPhBRF

hB"

tRNA-type

SNAP c

PSE TATA

TBPhBRFU

hB"

U6

pol lll

pol ll

A

B

TAF II s

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OCTAPOUH

POUS

45

19 43

50

190

Q

PSE

OIR

TATA

TBP

190

50

19 43TATA

TBP

Q

PSE

45

OIR

A

B

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Nouria HernandezsnRNA genes: a model system to study fundamental mechanisms of transcription

published online June 4, 2001J. Biol. Chem. 

  10.1074/jbc.R100032200Access the most updated version of this article at doi:

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  When a correction for this article is posted• 

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