Characterisation of human genomic sequences encoding RPB11-related proteins a and b
In addition to the previously characterised hRPB11
cDNA, referred to as hRPB11a
in the present work, a series of highly related human cDNAs were found in the databases ([24
], Table ). We show that these cDNAs were transcribed from a family of genomic sequences.
Accession numbers of RPB11 sequences
The screening of our genomic DNA library yielded several clones. Analysis of lambda clone 27 (Fig. ), revealed four coding exons within a 5.5 kb DNA sequence that we named hRPB11a gene, according to their identity with the hRPB11a cDNA. Lambda clone 11 was distinct from hRPB11a. Three exons were identified by their strong homology with exons 1, 2 and 3 from hRPB11a (Fig. , Table ). The fourth exon was identified by comparing this genomic sequence with two cDNAs from the database (Table ). This exon 4 sequence was specific to a subset of genomic sequences that we referred to as type b. hRPB11a and b genomic sequences diverged within intron 3 (Fig. ).
Figure 1 Structure of hRPB11 genes, mRNAs and proteins.A) Comparison of the structures of hRPB11a and b genomic sequences. Horizontal lines represent the human genomic sequences. The identified exons are indicated by boxes. The conserved 5' sequences encompassing (more ...)
Differential splicing of hRPB11b transcripts
We characterised two types of cDNAs from HeLa cells corresponding to hRPB11b transcripts and differing by the presence or absence of exon 3: they were named hRPB11bα and hRPB11bβ, respectively (Fig. , Table ). The absence of exon 3 switches the reading frame of exon 4, thereby extending the coding sequence (CDS) of hRPB11bβ into an additional exon 5, identified in another genomic sequence (Ac N° AC004951).
Most of the human cDNAs and ESTs in the databases (Table ) perfectly matched the cDNAs reconstituted from the exons of both hRPB11a and b genes, indicating that these sequences are transcribed in vivo. Exon 3 being present in all the genomic clones, we conclude that the hRPB11bβ cDNA is produced by differential splicing resulting in exon 3 skipping.
Three types of proteins are encoded by the hRPB11 genes
gene yields one type of mRNA that encodes the hRPB11a protein which was previously identified as a subunit of the human RPB complexes in Western-blots of immunoprecipitated RPB ([26
] and our unpublished data). We have presently identified two additional cDNAs, hRPB11bα
as distinct members of the same family.
Strikingly, as predicted from their sequences, the hRPB11a, bα and bβ polypeptides have similar sizes: 117, 115 and 116 residues, with calculated M.W. of 13.3, 13, 12.7 kDa, respectively (Fig. ). The N-terminal part of hRPB11a subunit differs only from the hRPB11b polypeptide by the presence of an additional Lys encoded at the junction between exons 1 and 2. By contrast, the C-terminal portions of these polypeptides differ drastically: while exon 4 of hRPB11a encodes a hydrophilic 11-residue peptide, it generates a rather hydrophobic 10-residue peptide in the case of hRPB11bα (Fig. ); concerning hRPB11bβ, due to exon 3 skipping, an unrelated peptide, rich in Pro (16%), Ala (14.5%), Gln (9%), His (9%) and Cys (7%) residues, is produced.
hRPB11 maps to three distinct loci on human chromosome 7
We localised the hRPB11 genomic sequences on metaphasic chromosomes with a fluorescent genomic probe encompassing the conserved exons 1 to 3 of hRPB11a (see Fig. ), thus revealing both hRPB11a and b genomic sequences. 50 metaphases were analysed: 90 % showed specific signals on chromosome 7, at positions q11.23 and q22, and about 80% at position p12.
A unique mRPB11 gene maps on mouse chromosome 5
The screening of our mouse genomic library yielded a unique mRPB11 gene (Fig. , Table ) which is transcribed into a unique type of transcript (Fig. , Table ) that encodes a mRPB11 protein identical to the human hRPB11a counterpart (Fig. ). In marked contrast to the human system, a single locus is detected on the murine chromosome 5, at cytogenetic band G (Fig. ).
Figure 2 Structure of mRPB11 gene, mRNA and protein.A) Comparison of the structures of hRPB11a and mRPB11 genomic sequences. Horizontal lines represent the genomic sequences. The identified exons are indicated by boxes. The conserved 5' sequences encompassing (more ...)
The hRPB11a and hRPB11b genomic sequences are transcribed in all human tissues tested
Expression of these cDNAs was tested in 16 independent human tissues by Northern-blot analysis (Fig. ). One major band was detected with each probe in all tissues. Strikingly, the relative levels of expression of hRPB11a versus hRPB11b isoforms varied, depending on the tissue. While hRPB11a was the major transcript in most tissues with highest levels in heart and skeletal muscle, hRPB11bα RNA was most abundant in the brain (note the different exposure times in Fig. ). hRPB11bβ transcripts were weak in all tissues, although more readily detected in the heart, skeletal muscle and ovary.
Figure 3 Nothern-blot analysis of hRPB11 expression.A) The hRPB11a, bα and bβ transcripts are represented as in Fig . The probes designed to reveal selectively each mRNA are indicated below in red. In the case of hRPB11b probes (more ...)
The proteins encoded by the three cDNAs exhibit specific interaction properties
The pairwise interaction abilities of all the hRPB subunits have previously been analysed using a GST pull-down assay [8
]. Similarly, we compared the interaction properties of hRPB11bα and bβ with those described for hRPB11a [24
] (Fig. ). In this assay, hRPB11a and bα revealed the ability to interact only with GST-hRPB3. By contrast, hRPB11bβ not only interacted with GST-hRPB3, but also with GST-hRPB1, 2, 4, 5, 6, 7 and 10β .
Figure 4 Interactions between hRPB11a, hRPB11bα and hRPB11bβ proteins with the twelve GST-hRPB subunits. Sf9 cells were coinfected with two recombinant baculoviruses, the first expressing one of the twelve GST-fused subunits or GST alone, the second (more ...)
Complementation experiments in budding yeast
We asked whether the human RPB11 homologues were able to compensate for the disruption of the Saccharomyces cerevisiae (Sc) essential RPB11 gene. In the complementation assay used, overexpression of ScRPB11 rescued this lethal phenotype by restoring yeast proliferation with a doubling time of 2 h (Fig. , line 1), whereas the empty vector did not (not shown). Under the conditions where all the human proteins were expressed to similar levels in the transformed yeast cells (data not shown), hRPB11a or bβ, did not rescue the ScRPB11 null allele (Fig. , lines 2 and 4). By contrast, hRPB11bα restored cell proliferation, although with a slower growth rate (Fig. , lines 3).
Figure 5 Complementation of rpb11::His3 yeast strains. The cDNAs assayed for complementation are listed on the left (lines 1–4). 30 cells of the complemented yeast strains were incubated at 28°C for 2 weeks on supplemented SD minimal medium, in (more ...)
hRPB11b genomic sequences share a domain with hPMS2L genes
Databases were screened for sequence similarities with the hRPB11b exons 4 and 5. The sequences of hRPB11bα and bβ, could be aligned with hPMS2L4 (Ac N° D38438) and hPMS2L13 (Ac N° AB017004): strikingly, the sequences of hRPB11b exon 4 and hPMS2L exon g were nearly identical (Fig. ). The hPMS2L cDNAs are encoded by a multigene family, in which exon g can be translated in two frames, depending on the gene (Fig. ). This is due to the presence of additional nucleotides at the 5' end of exon g, i.e. two A residues in hPMS2L13, when compared to hPMS2L4. Hence, very similar peptides can be produced from hPMS2L and hRPB11b cDNAs by completely distinct mechanisms involving small insertions and alternative splicing, respectively.
Figure 6 Similar peptides are produced via independent mechanisms in hRPB11b, hPMS2L4 and hPMS2L13 transcripts. The mRNAs are depicted by boxes that represent the exons. The black and grey areas represent the sequences that are specific to hRPB11b and hPMS2L, (more ...)