The mouse histone H2a gene contains a small element that facilitates cytoplasmic accumulation of intronless gene transcripts and of unspliced HIV-1-related mRNAs

Abstract

Histone mRNAs are naturally intronless and accumulate efficiently in the cytoplasm. To learn whether there are cis-acting sequences within histone genes that allow efficient cytoplasmic accumulation of RNAs, we made recombinant constructs in which sequences from the mouse H2a gene were cloned into a human beta-globin cDNA. By using transient transfection and RNase protection analysis, we demonstrate here that a 100-bp sequence within the H2a coding region permits efficient cytoplasmic accumulation of the globin cDNA transcripts. We also show that this sequence appears to suppress splicing and can functionally replace Rev and the Rev-responsive element in the cytoplasmic accumulation of unspliced HIV-1-related mRNAs. Like the Rev-responsive element, this sequence acts in an orientation-dependent manner. We thus propose that the sequence identified here may be a member of the cis-acting elements that facilitate the cytoplasmic accumulation of naturally intronless gene transcripts.

Figure 1

Polyadenylated H2a mRNA accumulates efficiently in the cytoplasm. (A) Schematic diagram of the plasmids. Shaded box represents the coding region of the H2a gene. Hatched boxes indicate either human or Xenopus β-globin genes. Arrows indicate transcriptional start sites. The thin line below the histone H2a construct marks the region used to build H-p(A). The numbers beneath the line represent nucleotides relative to the transcription start site. The probes used in RNase protection assays and the protected bands are indicated. The tilted portions of the probes depict nonhomologous sequences derived from plasmid vectors. In pβ1(−)2(−) the unique NcoI site is marked. The control construct H-XβG is described in Materials and Methods. Sizes are not to scale. (B) Autoradiograms of RNase protection assays of the globin and histone RNAs expressed in cells transfected with the indicated plasmids. M, molecular size markers (501, 404, 353, and 242 bp, respectively, from the top); c, cytoplasmic RNA; n, nuclear RNA; histone, mRNA from H-p(A); globin, RNA from pβ1(−)2(−); control, RNA from the cotransfected control plasmid pH-XβG. rel. cyto. accum., relative cytoplasmic mRNA accumulation obtained by setting the amount of cytoplasmic level of the globin RNA (lane 4) to 1, with normalization to the internal control RNA. C/N ratio, cytoplasmic and nuclear RNA distribution ratio after being normalized using the internal control RNA.

Figure 2

Sequences contained within the H2a gene facilitate the cytoplasmic accumulation of cDNA transcripts. (A) Structure of construct H-βG. The 496-bp histone fragment inserted at the unique NcoI site is shown, with its orientation indicated. Other symbols are the same as those described for Fig. 1A. (B) Results from an RNase protection assay of the globin and the histone-globin chimeric RNAs accumulated in the cells transfected with the indicated constructs. control, same as for Fig. 1B; globin, RNAs from pβ1(−)2(−) (lanes 1 and 2) and H-βG (lanes 3 and 4), respectively. rel. cyto. accum., relative cytoplasmic globin-related mRNA accumulation obtained as described in the legend to Fig. 1 after setting the amount of cytoplasmic level of the globin RNA (lane 1) to 1, with normalization to the internal control RNA. C/N ratio, cytoplasmic and nuclear RNA distribution ratio after being normalized using the internal control RNA.

Figure 3

A small element contained within the H2a coding region is sufficient to promote the cytoplasmic accumulation of the globin cDNA transcripts. (A) Structures of the plasmid constructs used. Shown at the top are the relative positions of the 100-bp (B) and 75-bp (N) fragments within the H2a gene. The numbers above or below indicate nucleotides relative to the transcription initiation site. Shown at the bottom are the three histone-globin chimeric gene constructs. The inserted fragments are indicated by thick lines, with arrows depicting their orientations. Other symbols are the same as for Fig. 1A. (B) Results from RNase protection assays of RNAs prepared from the cells transfected with the indicated plasmids. control, same as in Fig. 1B; globin, mRNAs from the indicated constructs. rel. cyto. accum., relative cytoplasmic globin mRNA accumulation obtained as described in the legend to Fig. 1 after arbitrarily setting the amount of cytoplasmic level of the globin RNA (lane 1) to 1, with normalization to the internal control RNA. C/N ratio, cytoplasmic and nuclear RNA distribution ratio after being normalized using the internal control RNA.

Figure 4

The histone element functionally mimics the Rev/RRE system. (A) Structures of the plasmid constructs. Shaded boxes and thin lines denote the exon and the intron sequences from the HIV-1 gene, respectively. The RRE sequence and the unique MscI site are also marked. In B-128 and Ba-128 the inserted histone fragments are indicated, with the arrows depicting the orientations. 5′ss, 5′-splice site; 3′ss, 3′-splice site; us, unspliced RNA; s, spliced RNA. (B) RNase protection assays of RNAs prepared from cells transfected with the indicated plasmids. +Rev, with cotransfection of the Rev-expressing vector; −Rev, without cotransfection of the Rev-expression vector. Other labels are identical to those for Fig. 1B. At the bottom is the quantitation of the fraction of either unspliced or spliced RNA that was detected in the cytoplasm of transfected cells, as well as the percentage of total RNA that was spliced.