(Received for publication, July 6, 1995; and in revised form, September 26, 1995)
From the
A subtractive cDNA cloning strategy was used to isolate a
1381-base pair human retina-specific cDNA, human retinal gene 4 (HRG4),
which hybridized to a 1.4-kilobase message in the retina and encoded a
240-amino acid acidic protein with a calculated molecular mass of
26,964 Da. The proximal of the conceptual protein sequence was rich in
glycine (18%) and proline (20%), had a predicted secondary structure of
turns, and showed a loose similarity (19-24%) to various
-collagen sequences, while the distal consisted of a mixture of
-helices,
-sheets, and turns. Genomic Southern analysis with
HRG4 showed cross-hybridizing sequences in six different species, and
HRG4 was 92% homologous with a 1264-base pair rat cDNA (rat retinal
gene 4; RRG4) at the protein level. The region of 100% identity between
the two sequences corresponded to the distal of the protein sequence
consisting of mixed secondary structures, suggesting a functionally
important domain. In vitro transcription and translation
corroborated the open reading frames corresponding to HRG4 and RRG4 in
the cDNAs. Expression of HRG4 in the retina was localized to the
photoreceptors by in situ hybridization. Developmentally, RRG4
began to be highly expressed around postnatal day 5 in the rat outer
retina when the photoreceptors begin to differentiate and rapidly
increased in expression to reach the mature adult level by postnatal
day 23. No diurnal fluctuation in expression of RRG4 was seen.
Isolation and characterization of mammalian genes specifically
or abundantly expressed in the retina have contributed greatly toward
the understanding of retinal biology and disease. Such genes include
rhodopsin, cGMP-phosphodiesterase, transducin, and arrestin that are
involved in
phototransduction(1, 2, 3, 4) ;
peripherin and rom-1 involved in photoreceptor disc structure
and function(5, 6) ; and interphotoreceptor retinol
binding protein involved in transport of retinol between the retinal
pigment epithelium (RPE) ()and photoreceptors(7) .
Candidate gene analyses of these genes in inherited retinal
degenerations have identified rhodopsin, peripherin,
cGMP-phosphodiesterase
, and rom-1 (possibly by itself
and in combination with peripherin) to be implicated in retinopathies
including retinitis pigmentosa and macular pattern
dystrophies(8, 9, 10, 11, 12, 13, 14, 15, 16) .
In order to expand our knowledge of retinal biology and to identify additional retinal genes that may be involved in retinal diseases, we have been isolating new retinal genes by a subtractive cDNA cloning strategy. This strategy has already resulted in the isolation and characterization of the cDNA and gene for human recoverin, a protein previously thought to be involved in the recovery of the depolarized state in phototransduction(17) , and X-arrestin, a new retinal arrestin that appears to play a desensitization role in a yet to be defined retina-specific signal transduction(18) . We now report another cDNA obtained by this strategy that represents a new retina-specific gene that is highly conserved in human and rat and that begins to express an acidic, hydrophilic protein with some similarity to collagen specifically in photoreceptors at the time of outer retinal maturation and continues to express this protein throughout adult life.
To study gene expression during retinal development, total retinal RNA was obtained from rats of various ages (postnatal day 0, 5, 7, 10, 15, 23, 30, 270, and 400). To avoid possible circadian variability in expression, all rats were sacrificed at about the same time of the day (4 p.m.). Northern blots with 8 µg of each RNA sample were hybridized with the full-length rat homologue cDNA (RRG4) or actin probe, washed at 52 °C, and autoradiographed as described above.
To examine possible diurnal
variations in expression, total retinal RNA was prepared from adult
rats at four different time points (7:00 a.m., 11:00 a.m., 7:00 p.m.,
and 11:00 p.m.). Rats were entrained to a cycle of 12 h of light
starting at 8:00 a.m. and 12 h of dark starting at 8:00 p.m.
Two-month-old rats from the same litter were divided into four groups,
and each group was sacrificed at a different time point. In the dark
period rats were killed and enucleated under dim red light, and retinas
were kept from bright light until completely dissolved in the guanidine
solution. Northern blots with 8 µg of each RNA sample were
hybridized with RRG4, S antigen or actin probe as described above. A
stretch of about 300 bp (1194-1482 bp) from the 3` end of a rat S
antigen cDNA (GenBank/EMBL, accession number X51781) was polymerase
chain reaction-amplified from a cDNA clone obtained from our rat
retinal cDNA library and used as a unique probe for S antigen. Final
washing was done with 0.1 SSC at 52 °C followed by
autoradiography.
Figure 1:
Northern blot
analysis of HRG4 in human tissues. Ten µg of total RNA from various
human tissues was applied in each lane: kidney (lane
1), lung (lane 2), fetal brain (lane 3), adult
brain (lane 4), skin fibroblast (lane 5), infant RPE (lane 6), adult RPE (lane 7), iris (lane 8),
cornea (lane 9), and retina (lane 10). A,
hybridized with P-labeled full-length cDNA probe of HRG4; arrowhead points to the transcript of approximately 1.4
kilobases seen only in the retina. B, hybridized with the
human
-actin probe to check on the quantity and quality of RNA
present in each lane. Positions of the 28 and 18 S ribosomal
RNA are shown.
The 1381-bp cDNA sequence contained an
open reading frame of 720 bp, beginning at the first ATG codon, coding
for a 240-amino acid protein with a pI of 5.96 and a calculated
molecular mass of 26,964 Da (Fig. 2). The conceptual protein
sequence showed a loose similarity to various collagen sequences
(29 out of the first 30 best matches) when analyzed by the FastDB
program (IntelliGenetics), although the matches were only in the range
of 19-24%. The region showing the matches was mainly the proximal
of the protein high in proline and glycine content. A typical
Gly-Xaa-Yaa repeat found in collagens, however, was not present in
HRG4. The Chou-Fasman secondary structure analysis (IntelliGenetics) of
the sequence demonstrated a stretch of turns in the glycine- and
proline-rich amino-terminal region of the protein, followed by
alternating
-helix,
-sheet, and turn structures (Fig. 3). The Kyte-Doolittle hydropathy plot indicated the HRG4
protein to be relatively hydrophilic, and a search for protein motifs
by the Quest program (IntelliGenetics) demonstrated a few frequently
occurring phosphorylation sites for casein kinase II, protein kinase C,
and tyrosine kinase. No signal sequence, transmembrane sequence, or
glycosylation site was recognized.
Figure 2: Nucleotide and deduced amino acid sequence of HRG4 cDNA. The putative translation initiation methionine and termination codons are underlined. The conceptual translation yields a 240-amino acid protein. The polyadenylation signal at nucleotide 1344 is overlined.
Figure 3:
The secondary structure of HRG4 predicted
by the Chou and Fasman algorithm(34) . Average propensity for
-helix,
-sheet, and turn was calculated for each segment of
the protein sequence, and the type of structure with the highest score
of propensity is listed beside the corresponding amino acid
residues.
Figure 4:
``Zoo'' Southern blot analysis.
Ten µg of genomic DNA from various species was digested with EcoRI, electrophoresed, and blotted. The blot was hybridized
with P-labeled full-length HRG4 cDNA probe. Lanes
1, mouse; lane 2, rabbit; lane 3, pig; lane
4, calf; lane 5, monkey; lane 6, human. Size
markers are HindIII-digested
DNA.
Figure 5: Nucleotide and deduced amino acid sequence of RRG4 cDNA. The putative translation initiation methionine and termination codons are underlined. The conceptual translation yields a 240-amino acid protein. The polyadenylation signal at nucleotide 1237 is overlined.
A comparison of the human and rat conceptual protein sequences showed them to be highly homologous, with an overall homology of 92% (Fig. 6). A distinct pattern of homology was present with a region of uniqueness in the first 50-60 residues rich in proline and glycine (67% homology) while the rest of the sequence was 100% identical between the human and rat proteins. This pattern of homology coincided with the distinct pattern of predicted secondary structure described above.
Figure 6: Alignment of HRG4 and RRG4. HRG4 (human) and RRG4 (rat) amino acid sequences were aligned by the GENALIGN program (GENALIGN is a copyrighted software product of IntelliGenetics, Inc.). Bars represent identical residues. A consensus sequence is shown at the bottom with uppercase letters indicating completely conserved residues.
The retina specificity of expression of the rat cDNA was confirmed by a Northern blot analysis of various rat tissue RNAs using the rat probe. Expression of a 1.3-kilobase RRG4 message was observed only in the retina among 10 different tissues analyzed (Fig. 7).
Figure 7:
Northern blot analysis of RRG4 in rat
tissues. Ten µg of total RNA from various rat tissues was applied
in each lane: lung (lane 1); liver (lane 2); kidney (lane 3); spleen (lane 4); brain (lane 5);
testis (lane 6); adrenal (lane 7); muscle (lane
8); heart (lane 9); retina (lane 10). A, hybridized with P-labeled full-length cDNA
probe of RRG4; the arrowhead points to the transcript of
approximately 1.3 kilobases seen only in the retina. B,
hybridized with human
-actin probe. Positions of the 28 and 18 S
ribosomal RNA are shown.
Figure 8:
In vitro transcription and
translation. A, the human cDNA clone was linearized with StuI in the 3` noncoding region, as indicated by the arrow, and transcribed into RNA using T3 RNA polymerase. The
rat clone was cut with StuI or MaeIII and transcribed
with T7 RNA polymerase. RNA transcripts were then translated in
vitro using the rabbit reticulocyte lysate with incorporation of
[S]methionine. Hatched boxes are the
open reading frames, and the numbers refer to their positions
from the first base of the cDNAs. B, autoradiograph of
SDS-polyacrylamide gel electrophoresis of the translation products with
different template RNAs. RNA templates made from human cDNA digested
with StuI (lane 1), rat cDNA digested with Mae III (lane 2), and rat cDNA digested with StuI (lane 3) are shown. Lane 4, no
RNA.
Figure 9: In situ hybridization of HRG4 in human retina. The result of liquid emulsion autoradiography of the retinal sections counterstained lightly with hematoxylin and eosin are shown. Hybridization was performed with antisense (A) or sense (B) riboprobe. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; IS, inner segment; OS, outer segment; RPE, retinal pigment epithelium. In addition to rods, some cones (arrowhead) show hybridized signals in their inner segments.
Figure 10: Developmental expression of RRG4 in rat retina. A, representative Northern blot analysis of RRG4 with 8 µg of total retinal RNAs obtained from rats of the indicated ages. Hybridization with RRG4 or actin probe is indicated by the lower and upper arrowheads, respectively. B, densitometric analysis of the expression level of RRG4. The intensity of the hybridized RRG4 message at each postnatal age was standardized with that of actin, and the ratio compared with the value at postnatal day 0 was plotted in logarithmic scale.
In agreement with the Northern analysis, in situ hybridization in the PND 0, 10, 30, and 270 rat retina demonstrated minimal signal at PND 0 but signal in the developing outer nuclear layer and the bacillary layer, corresponding to the developing inner and outer segments, at PND 10 (Fig. 11). The hybridization signal was present in the outer nuclear layer and inner segment at PND 30, and this pattern was unchanged at PND 270.
Figure 11: Developmental in situ hybridization of RRG4 in rat retina. Retinal sections from rats of the indicated ages were hybridized with antisense (A) or sense (B) riboprobe of RRG4 and subjected to liquid emulsion autoradiography and counterstaining with hematoxylin and eosin. GCL, ganglion cell layer; NBL, neuroblastic layer; RPE, retinal pigment epithelium; INL, inner nuclear layer; ONL, outer nuclear layer; BL, bacillary layer; IS, inner segment; OS, outer segment.
Figure 12:
Diurnal profile of RRG4 and S antigen
expression. A Northern blot with 8 µg each of total retinal RNA
samples prepared at the four indicated time points was hybridized with
RRG4 (A), S antigen (B), and actin (C)
probes. D, densitometric analysis of the expression level of
RRG4 () and S antigen (
). The intensity of the RRG4 and S
antigen messages at each time point was plotted as the percentage of
the 11:00 value after normalization with
actin.
We have isolated a new retina-specific cDNA (HRG4/RRG4) using
a previously described subtractive cDNA cloning approach(17) .
The encoded protein sequence showed an interesting two-domain structure
consisting of a proximal 50-60amino acid region rich in glycine
and proline and forming turns and a distal 180-190-residue region
made up of -helices,
-sheets, and turns. Although classical
collagen-associated Gly-Xaa-Yaa repeats were not present, the glycine-
and proline-rich region contributed to a loose similarity to various
-collagen sequences upon sequence matching.
The high degree of conservation between the human and the rat
sequence suggests the importance of structural conservation for
function of this retinal protein in different species. Moreover, the
pattern of homology between the two protein sequences seemed to point
to a functional basis of the putative two-domain structure described
above. Whereas the glycine and proline-rich amino-terminal region
consisting of 50-60 residues showed only 67% homology between the
two sequences, the rest of the molecule, made up of a mixture of
-helices,
-sheets, and turns, was 100% identical. Its 100%
conservation in human and rat seemed to indicate that this is the part
of the protein strictly conserved structurally.
The open reading frame sequences present in the human and rat cDNA clones were the longest stretches of such sequence present and contained putative translation initiation codons satisfying the consensus sequence described for initiation codons(29) . The in vitro transcription and translation, however, clearly indicated the functionality of the putative coding sequences in these clones. The 35-kDa HRG4 and 33-kDa RRG4 proteins could only be made from the described open reading frame sequences in the clones. The sizes of the translation products in the SDS-polyacrylamide gel electrophoresis analysis were larger than the calculated sizes (27 kDa versus 35 and 33 kDa), but the larger than expected size was seen even for the rat clone that was linearized at only 44 bases downstream of the termination codon. Since the larger size cannot be explained even if there was a read-through to the linearized end in the rat clone (28 kDa expected) and post-translational modification does not occur in rabbit reticulocyte system (confirmed by Stratagene), the size discrepancy appears to be due to the primary characteristic of the proteins. It may be due to the proline-rich feature of these sequences, a phenomenon observed for other proteins(30, 31) . Of course, the native proteins may have still a different size due to post-translational modification.
In situ hybridization analysis of HRG4 transcripts narrowed down the site of expression of this gene to the photoreceptors in the retina. Both rod and cone photoreceptors appeared to be expressing this gene. The developmental profile of expression of this gene demonstrated that it begins to be significantly expressed at around PND 5 in the developing rat retina when the photoreceptor differentiation begins to take place with the formation of the outer plexiform layer, outer nuclear layer, and the inner and outer segments. The expression level increased rapidly throughout the remainder of the period of photoreceptor maturation and reached the maximal stable level by PND 23, when the retina was fully developed. The level remained the same thereafter. Throughout the developmental stage and adulthood, the site of expression was in the photoreceptors. This developmental pattern of expression is identical to that of other photoreceptor-specific genes such as rhodopsin(32) , suggesting that HRG4/RRG4 may play a role in mature photoreceptors.
The function of the new photoreceptor-specific gene HRG4 cannot be clearly determined at this time. The hydrophilic nature of the encoded gene product suggests that it could be a soluble phototransduction protein, but it does not contain any recognizable sequence motif for such a protein, nor does its expression show fluctuation with the light/dark cycle, as has been shown for a number of phototransduction proteins including opsin, transducin, and S antigen(27, 28) . This may argue against this gene product being involved in phototransduction. Given the stable expression pattern during the 12:12 light/dark cycle and its loose similarity to collagen at the primary sequence and secondary structural levels, an attractive possibility is that HRG4 may be a structural protein or a matrix component. The interphotoreceptor matrix between the retinal pigment epithelium and photoreceptors has been described as not containing collagen(33) ; however, HRG4 may be a collagen-like protein in the interphotoreceptor matrix. Alternatively, HRG4 might be a structural or matrix protein of the photoreceptor synapse. Production of antibody and precise immunolocalization should clarify this question. Although its function has yet to be determined, the high degree of conservation of this gene in human and rat, its strict photoreceptor-specificity, and its distinct pattern of expression, beginning with the differentiation of the photoreceptors and continuing throughout life, clearly suggest that HRG4 may be an important retinal gene.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U40998 [GenBank]and U40999[GenBank].