Vol 13, Issue 5, 800-812, May 2003
LETTER
Retroposed Copies of the HMG Genes: A Window to Genome Dynamics
Liora Z. Strichman-Almashanu1,
Michael Bustin2 and
David Landsman1,3
1Computational Biology Branch, National Center for
Biotechnology Information, National Library of Medicine, National
Institutes of Health, Bethesda, Maryland 20894, USA;2
National Cancer Institute, National Institutes of Health,
Bethesda, Maryland 20892, USA
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ABSTRACT
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Retroposed copies (RPCs) of genes are functional (intronless
paralogs) or nonfunctional (processed pseudogenes) copies derived from
mRNA through a process of retrotransposition. Previous studies found
that gene families involved in mRNA translation or nuclear function
were more likely to have large numbers of RPCs. Here we characterize
RPCs of the few families coding for the abundant high-mobility-group
(HMG) proteins in humans. Using an algorithm we developed, we
identified and studied 219 HMG RPCs. For slightly more than 10% of
these RPCs, we found evidence indicating expression. Furthermore, eight
of these are potentially new members of the HMG families of proteins.
For three RPCs, the evidence indicated expression as part of other
transcripts; in all of these, we found the presence of alternative
splicing or multiple polyadenylation signals. RPC distribution among
the HMGs was not even, with 33–65 each for HMGB1, HMGB3, HMGN1, and
HMGN2, and 0–6 each for HMGA1, HMGA2, HMGB2, and HMGN3. Analysis of
the sequences flanking the RPCs revealed that the junction between the
target site duplications and the 5'-flanking sequences exhibited the
same TT/AAAA consensus found for the L1 endonuclease, supporting an
L1-mediated retrotransposition mechanism. Finally, because our
algorithm included aligning RPC flanking sequences with the
corresponding HMG genomic sequence, we were able to identify
transcribed regions of HMG genes that were not part of the published
mRNA sequences.
Processed pseudogenes (PPSs) are nonfunctional
genomic insertions that arise by a process of retrotransposition. This
process involves reverse transcription of processed RNA polymerase II
transcripts and integration into a random site in the genome (Vanin
1985 ; Weiner et al. 1986; Maestre et al. 1995). The resulting
insertions have a structure similar to that of retroposons such as Alu
and L1 elements in that they lack introns, have DNA-encoded 3' poly(A)
tails, and direct repeat sequences flanking the insertion, termed
target site duplications or TSDs. These similarities between Alu and L1
elements led to the hypothesis that Alu element retrotransposition is
mediated by the L1 reverse transcriptase (Boeke 1997). It has
subsequently been shown that the length and composition of Alu element
TSDs fit the L1 reverse transcriptase preference (Jurka 1997).
Following the same logic, PPSs were also thought to be retroposed via
the L1 reverse transcriptase machinery, a process that was carried out
successfully in cultured cells (Esnault et al. 2000). However, the
features of the TSDs and structure of the insertions were only studied
in isolated cases.
Because the inserted sequences are promoterless and nonfunctional, they
accumulate insertions, deletions, and single nucleotide substitutions
that lead to frame shifts and premature stop codons (Ophir and Graur
1997). However, if the insertion site happens to be downstream from a
promoter, the inserted sequence may become expressed (Rogalla et al.
2000; Birger et al. 2001). For this reason we prefer to use the term
"retroposed copies" (RPCs) rather than "processed pseudogenes."
Earlier studies (Goncalves et al. 2000; Venter et al. 2001; Harrison et
al. 2002) found that genes giving rise to RPCs generally have short
transcripts, are widely expressed, GC-poor, and have nuclear or protein
synthesis functions. A recent study (Zhang et al. 2002) analyzed RPCs
of the ribosomal proteins genes and found them to be uniformly
distributed throughout the genome, with a preference toward
GC-intermediate regions. This analysis also found GC-poor genes to
produce more RPCs. Another group of genes with a predicted large number
of RPCs encodes the HMG (high-mobility group) proteins. These are
abundant nonhistone nuclear proteins that bind to DNA or chromatin and
affect DNA-related activities such as replication, transcription, and
chromatin compaction (for review, see Bustin 1999). The HMGs are
divided into three families: HMGB genes, which contain an HMG-box
domain that bends DNA, affecting transcription, and include the genes
HMGB1, HMGB2, and HMGB3 (formerly known as HMG1, HMG2, and HMG4,
respectively); HMGN genes, which contain a nucleosomal binding domain
affecting replication, chromatin structure, and transcription, and
include HMGN1, HMGN2, and HMGN3 (formerly HMG14, HMG17, and Trip7,
respectively); and HMGA genes, which contain an AT-hook domain
affecting chromatin structure and transcription, and include the splice
isoforms HMGA1a/b/c/, and HMGA2 (formerly HMGI, HMGY, HMGR, and HMGC,
respectively, summarized in the HMG chromosomal proteins nomenclature
home page at
http://www.informatics.jax.org/mgihome/nomen/genefamilies/hmgfamily.shtml).
HMG genes in both human and mouse had been suspected to have a large
number of RPCs by Southern blot analyses (Landsman et al. 1986a,b;
Srikantha et al. 1987; Johnson et al. 1988, 1989, 1992; Wen et al.
1989), and several HMG RPCs were identified and mapped in human
(Srikantha et al. 1987; Stros and Dixon 1993; Rogalla et al. 1998,
2001; Dunham et al. 1999). Two cases of expressed HMG RPCs have been
described. In one, the expressed sequence was mostly similar to the
original HMG mRNA, with a similar open reading frame (ORF), and most
likely a similar function (Birger et al. 2001). In the other, the
retroposed copy sequence was expressed as an alternative exon of the
SP100 gene, adding the HMGB DNA-binding domain to the SP100 nuclear
antigen (Rogalla et al. 2000).
Retroposed copies of known genes present us with a unique tool to
investigate genome dynamics and function, to learn about the process of
retrotransposition, to use as complementary information to ESTs and
cDNAs by representing a variety of transcripts that may or may not be
represented in the present expression databases, and to learn about the
creation of new genes. The anticipated large number of RPCs for the HMG
genes in whole genome studies provided us with the data set for these
analyses.
Previous whole genome studies of RPCs focused mainly on types of genes
that were likely to generate them, excluded potentially expressed RPCs,
and used criteria too general for an in-depth analysis (Goncalves et
al. 2000; Venter et al. 2001). We devised a strategy aimed at the
specific and accurate identification of HMG RPCs and their flanking
sequences. We describe their genomic distribution, their flanking
sequence characteristics, and their potential for being expressed, and
we identify sequences included in RPCs that are not represented in the
corresponding known mRNA sequences.
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RESULTS
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Retroposed Copies (RPCs) of HMG Genes
We used eight human HMG mRNA sequences representing HMGA1, HMGA2,
HMGB1, HMGB2, HMGB3, HMGN1, HMGN2, and HMGN3 (Fig.
1; Table 1) in a
BLASTN search (Altschul et al. 1990) against the human chromosome
contig database at NCBI, which contains draft and finished sequence
contigs that are part of the RefSeq database (Pruitt and Maglott 2001).
In the first round, we classified genomic fragments corresponding to
HMG parent genes as hits with  97% identities to the HMG mRNA
sequence, and covering 70% of its length. We additionally required
that an overlap or gap in the mRNA sequence between local alignments
will be  4 bp, so these local alignments would correspond to exons,
and the location of the edges of local alignments in the mRNA would
correspond to splice junctions. RPCs were subsequently defined as hits
in which the alignments with the HMG mRNA were uninterrupted through at
least one identified splice junction, as defined above, evidence that
splicing had occurred (Fig. 1). We identified 219 RPCs with identity to
the HMG mRNA sequences ranging from 64% to 98%; 78% of them had
70% coverage of the mRNA (Table 1). An additional 37 hits were all
confined to the last exon of the HMG gene, and as these hits did not
cross splice junctions, we categorized them as PSs (for pseudogenes).
Although they were not RPCs by our criteria, some had TSDs, and it is
likely that these hits represented truncated RPCs resulting from an
incomplete reverse transcription event. The PS hits were not included
in the analyses of chromosomes, TSDs, repeats and GC content in
flanking sequence, and insertion start/end points; however, we did
analyze them individually. RPCs could be divided into two groups. The
abundant group included RPCs of HMGB1, HMGB3, HMGN1, and HMGN2, which
each had 33–65 copies; the sparse group included RPCs of HMGA1, HMGA2,
HMGB2, and HMGN3, with each only 0–6 copies. Among the RPCs were the
previously reported processed pseudogenes HMG1L1/3/4/5/6/8/9 (Stros and
Dixon 1993; Rogalla et al. 1998), HMG17L1/3 (Dunham et al. 1999),
clones 28H and 60H (Srikantha et al. 1987), and HMGIYL3 (Rogalla et al.
2001).
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Figure 1. Identification of retroposed copies (RPCs) for HMG genes. HMG mRNA
sequences (in gray) were soft-masked and used in BLAST searches against
the Human Genome Build #26 chromosome files (in blue). HMG parental
genes were first identified (see Methods), and splice junction
positions in the mRNA sequences were defined from these alignments
(black arrowheads) and were subsequently used as guides for identifying
RPCs (in red). RPC endpoints were adjusted by conducting a BLAST2SEQ
comparison against the corresponding HMG gene, as well as with
less-stringent parameters (see Methods) against the HMG mRNA.
Horizontal dashed lines represent gaps in the alignment, and vertical
dashed lines represent the splice positions in the mRNA–RPC
alignment.
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Characterization of Integration Sites
An analysis of pseudogene distribution on Chromosomes 21 and 22
indicated an uneven distribution across the chromosome length, with an
excess of pseudogenes in the 5 Mb closest to the centromere (Harrison
et al. 2002). To find potential hot spots for RPC integration, we
looked at their chromosomal localization (Fig.
2). Although the RPCs seemed to be
distributed throughout the genome, a region of higher density was found
on Chromosome arm 15q. Conversely, no RPCs were found on Chromosome Y,
and Chromosome 8 contained only HMGB1 RPCs. We did not find, however,
an increased density of RPCs around centromeres.
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Figure 2. Genomic distribution of 198 mapped RPCs. The colored tick marks
represent individual RPC locations according to the legend; a diamond
shape represents the corresponding gene. The chromosome shading
illustrates the number of RPCs in 20 megabase bins, according to the
horizontal scale below; red regions in the chromosomes are
centromeres.
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To scan the integration sites for repeats and unique sequences, we used
RepeatMasker
(http://repeatmasker.genome.washington.edu/cgi-bin/RepeatMasker;
A.F.A. Smit and P. Green, unpubl.) to identify and mask repetitive
elements in the 250 bp on either side of the RPCs, followed by BLASTN
searches with the masked sequences. Forty-five percent of the combined
5'-flanking sequence and 39% of the combined 3'-flanking sequence were
masked by RepeatMasker and labeled as interspersed repeats (Table
2). In comparison, 45% of the human genome
is composed of interspersed repeats (Lander et al. 2001). Most of the
difference between the 5' and 3' flanks was caused by an excess of SINE
elements (specifically Alu elements) in the 5'-flanking sequence (18%)
relative to the 3'-flanking sequence (13%). The genomic fraction of
SINE elements is also 13%. This could be a result of the preference of
RPCs to integrate in stretches of A, which occur more frequently in the
3' ends of Alu elements.
Four RPCs were found to flank a gene or another putative RPC (Fig.
3). An HMGN1 RPC on Chromosome 10p13 was
identified in a tail-to-tail configuration with a sequence 92%
identical to the 3' end of Ca-ATPase (M23114) mRNA located on 12q24
(Fig. 3A). The corresponding genomic sequence for Ca-ATPase did not
contain introns, and we could not identify a poly(A) tract or a target
site duplication to ascertain whether it was, indeed, an RPC. From the
differences in similarity to their respective mRNAs located on separate
chromosomes, as well as from their relative orientation, it is most
likely that these two pseudogenes were generated in two separate
events. An HMGN1 RPC on 2q23 overlaps the 5' end of the KARP-1 gene
(AF039597) in the opposite orientation, and encodes the first two exons
of this gene, according to the GenBank gene record (Fig. 3B). An HMGN2
RPC on 2q13 was identified in intron 4 of the IL1 homolog (NM_019618),
in the opposite orientation (Fig. 3C). An HMGN2 RPC on 17q23 was
identified immediately upstream of a sequence 95% identical to
ribosomal protein L12 (L06505) and containing a poly(A) tail and TSD,
most likely also an RPC (Fig. 3D). It is unlikely for both of these
RPCs to have been created in a single event because both contained
distinguishable TSDs derived from the sequence between them, which was
identified by RepeatMasker to be a fragment from a THE1-int LTR/MaLR
element (see Fig. 3D).
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Figure 3. Genes and pseudogenes flanking HMG RPCs. HMG RPC (shown as red boxes)
flanking sequences were masked and used in a BLAST search to find
surrounding genes and pseudogenes (depicted as blue boxes). The
orientation of all HMG RPCs is left to right.
(A) A pseudogene of the Ca-ATPase (M23114, 92% identical) was
found in the reverse orientation (denoted by a thin blue arrow) to an
HMGN1 RPC (89% identical), at a distance of 40 bp (intervening black
line). (B) An HMGN1 RPC (88% identical) in the 5' flank of
the KARP-1 gene contributing two exons. The three hatched boxes depict
Alu elements. (C) An HMGN2 RPC (89% identical) in the fourth
intron of the IL-1 homolog. (D) An HMGN2 RPC (93% identical)
upstream and in the same orientation as an RPC of ribosomal protein L12
(95% identical; see text). The 47 bp between them is a fragment of a
THE1 element (underlined in the middle insert), from which the TSDs for
both RPCs are derived (colored nucleotides). (Open triangles) TSDs; TSD
sequences appear in the inserts, and nucleotides in green represent
ambiguity; (AAA) poly(A) tracts; (black arrowheads) splice sites;
(hatched boxes) repetitive elements; (thick blue arrows) CDS; (dotted
lines) position of introns in CDS; (lines between the boxes)
intervening genomic DNA, or introns if colored; (–//–) a break
artificially inserted into the long sequence for convenient display.
Figure not drawn to scale.
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The base composition was calculated for 500 bp flanking the RPCs and
was found to be AT-rich (Table 2). The 3'-flanking sequence average GC
content was 37%, and the 5'-flanking sequence average GC content was
40%, both close to the genomic average of 41% (Lander et al. 2001).
The slight decrease in GC content in the 3' flank could be due to the
presence of poly(A) tracts.
Expression of RPCs
Most RPCs are not expressed, and therefore are referred to as
processed pseudogenes. However, the few that are expressed can
potentially give rise to novel HMG genes, or to modular combinations of
HMG domains with domains from other genes. We identified potentially
expressed RPCs through BLASTN searches against mRNA and EST databases.
A significant EST or mRNA hit was considered evidence for potential
expression if it exhibited 99% similarity to the RPC but 97% to
the HMG mRNA.
Altogether, we found 22 potentially expressed RPCs and 3 potentially
expressed PSs (Table 3), of which three had
already been reported: HMG1L3, an RPC of HMGB1 that is expressed as an
alternative exon of the SP100 gene (Rogalla et al. 2000); HMGN4, an
intronless gene that consists of an RPC of HMGN2 surrounded by
additional sequence (Birger et al. 2001); and HMG17L1, an RPC of HMGN2
reported as part of the Chromosome 22 project (Dunham et al. 1999).
Of the 22 novel expressed RPCs and PSs, 6 had an ORF similar to the
corresponding HMG ORF (e.g., Fig. 4A,C,E);
6 had an ORF unrelated to the HMG ORF—a different reading frame,
orientation, or location in the HMG transcript (e.g., Fig. 4C,D); and 3
were included as part of a transcript with a coding sequence (CDS)
outside the pseudogene region (e.g., Fig. 4B,D,F). Interestingly, two
of the three encoded alternative exons (Fig. 4B,D), and one was
embedded in an alternative 3'-UTR region, thereby supplying an
alternative polyadenylation signal. This last gene model was supported
by human ESTs, as well as by mRNAs from monkey and mouse (Fig. 4F).
Alternative splicing also plays a role in the only published example of
an HMG RPC expressed as part of another gene, in which case an RPC of
HMGB1 encodes an alternative exon of the SP100 gene, donating an HMG
domain to the SP100 nuclear antigen to create the SP100–HMG splice
variant (Rogalla et al. 2000). Six potentially expressed pseudogenes
included putative splice sites (e.g., Fig. 4B,D,E), which was supported
by the alignment of ESTs with the genomic sequence in some cases.
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Figure 4. Potentially expressed RPCs. RPCs were masked and used in a BLAST search
against the mRNA and EST databases. (A) HMGA1 RPC lacking exon
2 (vertical line) and with a 33-bp insertion (stippled box) harbors an
ORF (red arrow) similar to HMGA1 CDS with an insertion (embedded
stippled line) and a premature stop codon (asterisk), as well as one
EST (represented by a thin black arrow). (Gray arrow) The CDS of HMGA1.
(B) An HMGA1 PS with a 500-bp deletion (vertical line) encodes
exons shared with an alternatively spliced mRNA (NM_052844; thin blue
arrow) through ESTs (thin black arrows); (dotted lines) introns in
transcribed sequence. The HMG insertion ends with poly(A); however, it
is internal to the HMGA1 transcript. (C) HMGB1 RPC has an ORF
(thick black arrow) in an opposite orientation to EST AW270920, and
another ORF (thick red arrow) similar to HMG CDS. (D) An HMGN1
RPC has an ORF within EST AL138207 (thick black arrow) that is not
similar to the HMGN1 CDS, and encodes an exon shared with an
alternatively spliced mRNA (NM_023071; thin blue arrow) through ESTs.
EST AA584308 is part of UniGene cluster Hs.152982. (E) An
HMGN2 RPC has an ORF similar to HMG CDS (thick red arrow) and three
ESTs in the same orientation. (F) An HMGN2 RPC in the 3'-UTR
of cDNA AB037814 provides an alternative poly(A) signal
(downward-pointing arrow). ESTs illustrate the use of both signals; a +
after EST BF055348 stands for more ESTs at the same position. The gene
structure of AB037814 was derived from an alignment with genomic DNA.
(Green arrows) Monkey and mouse mRNA sequences similar to AB037814 that
do not include an HMG sequence; the black part of the mouse DNA
represents nonaligning sequence; (hatched box) a sequence of repetitive
elements; (open triangles) TSDs, TSD sequences appear in the inserts,
nucleotides in green represent ambiguity; (AAA) poly(A) tracts;
(downward-pointing arrowheads) poly(A) signals; (upward-pointing
arrowheads) splice sites; (thick arrows) ORFs or CDSs: The position of
the HMG CDS is depicted in gray; ORFs similar to HMG CDS are red; ORFs
not similar to HMG CDS are black, and CDSs outside the RPC region are
blue. (Dotted lines) Intron positions in transcripts; (lines between
the boxes) introns in genomic DNA; 3' EST orientation is reversed to
presumed sense; not drawn to scale.
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From RPCs Back to mRNA
Because RPCs are derived from mRNA molecules, a comparative analysis
of their sequences may reveal the existence of variant mRNA species
that are expressed only under specific conditions, are extinct, or are
difficult to clone. Through our survey of RPCs derived from HMG mRNAs,
we found RPCs that contained sequences present in the genomic sequence
of the HMG gene but not in the published mRNA databases (Table
4).
The alignment of six HMGB1 RPCs from five different chromosomes to the
HMGB1 genomic sequence did not end at the known 3' end of the gene, but
rather extended downstream for an additional 1 kb, including a putative
polyadenylation signal (Fig. 5; Table 4).
This is evidence that a transcript longer than the known HMGB1 mRNA was
being created through the alternative use of poly(A) signals. The
percent identity of these RPCs to HMGB1 ranged from 82% to 97%,
implying that they were created in separate retrotransposition events
and that this longer transcript represented  10% of HMGB1
transcripts. The existence of this 3'-extended HMGB1 transcript was
supported by several human ESTs and a cDNA clone from spleen
(AK057120). Additionally, we found 40 RPCs of HMGB1 that extended up to
100 bp upstream of the mRNA start point (Fig. 5; Table 4), indicating a
previously inaccurate identification of the correct transcription start
site for this gene. Such 5'- or 3'-extended RPCs were found for other
HMGs as well, in many cases without a corresponding transcript in
expression databases (summarized in Table 4).
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Figure 5. Transcripts of the human HMGB1 gene: alternative 3' end, extended 5'
end. RPCs aligned directly to the HMGB1 genomic DNA (gray boxes) were
found to have sequences extending upstream and downstream (black and
blue boxes) relative to the HMGB1 RefSeq mRNA NM_002128. The middle
structure is a schematic representing all RPCs derived from HMGB1, with
gray structures corresponding to NM_002128 sequence, blue structures
corresponding to a cDNA downstream from NM_002128 (AL110194), and black
boxes are genomic sequences outside these cDNAs. The numbers within the
boxes show how many RPCs start (with upward-pointing red arrows) or end
(with downward-pointing red arrows) within this region. ESTs shown
belong to UniGene cluster Hs.337757, and illustrate the alternative use
of poly(A) signals (downward-pointing blue and gray arrowheads), as
well as the existence of transcription upstream and downstream to
NM_002128, also supported by a cDNA (AK057120). (Green arrows) Pig and
mouse HMGB1 mRNAs, which also extend downstream of NM_002128. (Open
triangles) TSDs; (AAA) poly(A) tracts; (upward-pointing black
arrowheads) splice sites. The dotted lines in AK057120 depict a 70-bp
deletion, and 3' EST orientations are reversed.
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Evidence for alternative splicing of HMG genes was observed for HMGA1,
which is known to have several alternative transcripts with the CDS
starting at exon 3 (Johnson et al. 1989; Friedmann et al. 1993). Six
RPCs were found for HMGA1 (Fig. 6). All of
these RPC sequences begin within the first exon and correspond to a
variety of spliced isoforms, which include HMGA1a (variant 2,
NM_002131); HMGA1b, which has an insertion of 33 bp from intron 3
adding 11 amino acids to the CDS (variant 1, X14957); and another
isoform that was the result of exon 2 skipping (variant 5, M23617).
None of the RPCs had the structure of HMGA1c (AF176039) or included the
alternative exons present in variants 3, 4, 6, or 7. A novel splice
isoform with no corresponding mRNA was represented among the RPCs,
resulting from a combination of exon 2 skipping and a 33-bp insertion.
This finding demonstrates the utility of this method, enabling us to
discover new variant transcripts.
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Figure 6. HMGA1 RPCs aligned with mRNA splice isoforms. (Black arrow in the
middle) The NM_002131.1 RefSeq mRNA; (thick black arrow) its CDS
position. Above are transcript variants in gray, below are RPCs in red;
different shades of blue lines represent different alternative exons,
and the hatched line in variant 6 represents an Alu element.
(Downward-pointing arrowheads) The position of an additional 33 bp
between exons 3 and 4 in some of the transcripts and RPCs.
(Upward-pointing arrowheads) Splice positions; (dotted lines) deletions
or exon skipping.
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Mechanism of Generation
It has been suggested that RPCs are generated by the L1 reverse
transcriptase (Ostertag and Kazazian Jr. 2001). In common with the end
product of retrotransposition by an L1 reverse transcriptase, RPCs
include a poly(A) tract at the 3' end of the insertion and are flanked
by direct repeats termed target site duplications (Vanin 1985). We
scanned all the RPC flanking sequences for direct repeats that were 7
bp long and 90% identical, as well as for potential poly(A) tracts
close to the 3' end of the insertion. The distance between the direct
repeats and the insertion ends was permitted to be up to 30 bp to allow
for differences resulting from mutations and inaccurate identification
of the ends. TSDs were found for 48% of all RPCs. However, a higher
fraction of TSDs was found for subgroups in which the ends were defined
with higher confidence. For example, 69% of RPCs that matched 70%
of the mRNA length had TSDs. Furthermore, more recently retroposed RPCs
that share a higher percentage of identity with an mRNA are more likely
to contain intact TSDs (data not shown).
The L1 reverse transcriptase had been shown to have a sequence
preference for integration (Feng et al. 1996). A corresponding
preference of TT/AAA was found at the junctions between 5' TSDs and
5'-flanking sequences of Alu elements that are thought to be retroposed
via the L1 reverse transcriptase machinery (Boeke 1997; Jurka 1997). To
find sequence patterns around the TSD–5'-flanking sequence junction
(which represents the first nick in the L1 integration process), we
aligned TSDs including 10 bp of 5'-flanking sequence justified at the
5' end. We also aligned TSDs with 10 bp of 3'-flanking sequence
justified at the 3' end; this junction between TSD and the 3'-flanking
sequence represents the second nick. The sequence preference found at
the junction between the 5' TSD and the 5'-flanking sequence was
TT/AAA, which corresponds to the L1 preference as well as to the
sequence at the 5' junction of Alu elements (Fig.
7A). No consensus could be found at the 3'
junction. It is possible that the second nick is determined by the
distance from the first nick rather than by the sequence at the site
(Jurka 1997; data not shown). This distance corresponds to the length
of the TSD, which is generally 15 bp. We found an average length of
14 bp for TSDs (Fig. 7B), as well as false peaks at the shortest TSD
size allowed (7 bp) and at 10 bp, the shortest in which mismatch is
allowed. The size of these false peaks is reduced when considering only
insertions with >90% identities to HMG or that cover >70% of its
length (data not shown).
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Figure 7. TSD composition, length distribution, and inverted/truncated elements.
(A) A sequence logo representation of the 5' flank junction
for TSDs 10 bp (n = 37) without ambiguities. The cartoon
underneath represents the junction position relative to the RPC.
(B) TSD length distribution of RPCs (n = 116).
(C) An HMGN1 RPC 5'-truncated at bp 152 and inverted at bp
299–316 (gi 16160227, bp 1,018,816–1,019,883). (D) An HMGN2
PS 5'-truncated at bp 788 and inverted at bp 938–959 (gi 16157330, bp
297,023–297,461). This sequence has a TSD and a poly(A) tract, most
likely an RPC; see text. Numbers correspond to position in the HMG
mRNA; (triangles) TSDs; (thin arrows) direction of corresponding HMG
transcript.
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Because inversions/truncations are observed in 12% of L1 elements and
are thought to be a feature of the L1 reverse transcriptase (Symer et
al. 2002; Szak et al. 2002), we searched for and identified similar
inversions/truncations in RPCs. One RPC and one PS were found to have
an inverted and truncated 5' region (Fig. 7 C,D), and because we could
find a TSD and a poly(A) tract for the PS, it strengthens the
likelihood that it was, indeed, a truncated RPC (Fig. 7D).
Taken together, the similarity of RPC structure to that of L1 and Alu
elements, the similar length distribution and sequence composition of
their TSDs, and the presence of inversions/truncations events among the
RPCs, all support an L1 reverse transcriptase mechanism for the
generation of RPCs. L1 is a mammalian retroelement—although all
mammals were found to have sequences related to L1 elements, no such
sequences could be found in nonmammals such as duck and sea urchin
(Hutchison et al. 1989). If L1 is, indeed, the agent retroposing RPCs,
it is not surprising that HMG RPCs could not be found in chicken
(Landsman et al. 1988; Srikantha et al. 1989; Lum et al. 2000).
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DISCUSSION
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RPC Data as Supplement for Expression Databases
Several studies and genomic surveys of retroposed copies of genes
have been carried out, focusing mainly on the characteristics of genes
that generate RPCs (Goncalves et al. 2000; Venter et al. 2001; Harrison
et al. 2002). In this work we chose a different perspective and studied
the insertions themselves to better characterize each RPC and gain
insight into the mechanism of their generation.
Because RPCs are derived from mRNAs, they provide a source of
transcript information that is independent of expression sequence
databases. RPCs reflect active transcription of genes at a certain time
and in a certain tissue, and are free of cloning artifacts. If aligned
directly to HMG genes, RPCs can reveal the usage of certain transcripts
or isoforms that no longer exist or are rarely expressed, as well as
sequences that have eluded cloning. We found deviations from published
mRNA sequences for several HMG genes. The most instructive example
comes from HMGB1. We found that 10% of HMGB1 RPCs with a wide range of
identities with HMGB1 mRNA included an additional 1 kb of sequence
immediately 3' to the end of the gene. EST sequences and one cDNA clone
isolated from spleen were found matching the extended sequence (Fig.
5), implying that this transcript presently exists. Because the known
HMGB1 mRNA, the RPCs, and the 3' ESTs starting from the respective
endpoints all ended with poly(A) tails, we concluded that this longer
transcript is the result of an alternative downstream polyadenylation
signal. Both the long and the short transcripts encode the same
protein, thus the additional sequence could serve a regulatory role.
The existence of alternative transcripts for HMGB1 of the sizes
corresponding to the known and the new transcription endpoint was
previously observed on a Northern blot (Wen et al. 1989).
Interestingly, this longer 3'-UTR was included in the only mRNA from
pig found in GenBank, and together with the shorter isoform in mouse,
indicating that it is evolutionarily conserved. At the 5' end of HMGB1,
66% of RPCs included additional sequence of up to 100 bp upstream.
ESTs were found to extend as far as 80 bp upstream, and a cDNA was
found that extended 40 bp upstream (Fig. 5; Table 4). RPCs of other
HMGs also included 5'-extended sequences that were not found in the
mRNA or EST databases (Table 4). Inaccurate 5'-end assignment of mRNA
is a known artifact of cDNA library production methods. The study of
RPCs circumvents this problem and enables an improved accuracy of
5'-end definition.
HMGA1 was the only HMG gene previously known to have alternatively
spliced isoforms, and although only six RPCs were found for this gene,
they represented three known spliced isoforms as well as at least one
that does not exist in GenBank (Fig. 6). This implies that there are
species of HMGA1 cDNAs that are not represented in expression
databases, and that a variety of HMGA1 isoforms is expressed in the
germ line.
The uneven distribution of RPCs among the HMG genes could be partly
explained by differences in expression levels. HMGB1/2 are the most
highly expressed followed by HMGN1/2, whereas the least expressed is
HMGA1 (Bustin 1999). Also, if we set aside HMGB3 and HMGN3, the three
shortest transcripts generated the most RPCs. Both expression levels
and transcript size were among the features previously found to
distinguish genes that produce RPCs (Goncalves et al. 2000; Venter et
al. 2001). However, these features alone do not explain why HMGB2
generated very few RPCs, HMGN1/2 generated more RPCs than HMGB1, and
the shortest transcript, HMGN3, only generated one RPC. In a recently
published analysis of the ribosomal protein RPCs (Zhang et al. 2002),
the authors find an inverse correlation between the GC content of the
CDS and the number of RPCs. However, no similar properties seem
apparent from our data (see Table 1).
One prerequisite for retrotransposition is expression in germ-line
cells, both because in this tissue L1 reverse transcriptase is
expressed (Ostertag and Kazazian Jr. 2001), and because germ-line
expression is essential for vertical transmission. Studies of a
transgenic mouse model showed that L1 expression occurs in late-meiotic
and post-meiotic male germ cells (Ostertag et al. 2000). On the other
hand, HMGB2 expression in mice testes was found to be restricted to
primary spermatocytes, which occur just prior to the first meiotic
prophase (Ronfani et al. 2001). This timing difference could explain
why there are so few HMGB2 RPCs relative to HMGB1, in spite of the high
similarity in structure and function between these two genes. HMGB3
(previously HMG4) has been shown to be expressed in early development
(Vaccari et al. 1998). However, from the abundance of HMGB3 RPCs, it is
most likely also expressed in germ-line cells. HMGN3 was found to be
expressed in testes (West et al. 2001), which does not explain why only
one RPC was found for this gene.
Another factor possibly determining the number of RPCs generated is the
location of the mRNA in the cell. It has been hypothesized that Alu
elements are successful retroposons in spite of their lack of coding
regions, because they are derived from the 7SL RNA gene, which is part
of the signal recognition particle localized to the endoplasmic
reticulum (Boeke 1997). Alu elements can bind proteins of the signal
recognition particle and thus be retained in the endoplasmic reticulum,
in position to be retroposed by the L1 reverse transcriptase (Mighell
et al. 1997).
Potentially Expressed RPCs Shed Light on Genome Evolution
Perhaps one of the most interesting contributions of our work is the
discovery of potentially expressed sequences. Two groups were
identified as most informative of the novel expressed RPCs. The first
group consisted of six RPCs with an ORF similar to that of the
corresponding canonical HMG (Table 3; Fig. 4). These could represent
potential intronless paralogs of the respective genes. Indeed, the
recently discovered HMGN4 gene is comprised mostly of an expressed RPC
of HMGN2 (Birger et al. 2001). The second group consisted of three
pseudogenes that were included as part of transcripts with a CDS from
non-HMG genes (Table 3; Fig. 4). Interestingly, these HMG exons were
all part of alternatively spliced transcripts or an altered poly(A)
signal usage. In the only published example of an HMG RPC expressed as
part of another gene, it was in an alternative exon of the SP100
nuclear antigen splice variant termed SP100-HMG, an HMG-box gene
(Rogalla et al. 2000). HMG-box genes, as opposed to the canonical HMG
genes, are genes that have other specificities or functions in addition
to the HMG-box (Landsman and Bustin 1993). Therefore,
retrotransposition could be one mechanism through which the HMG-box
proteins are generated. This second group of transcribed RPCs is
especially interesting because it can potentially give rise to new
genes or domain combinations. The significance of the new exons being
part of alternative transcripts may be that insertion into a gene can
disrupt its function and be selected against, whereas expression as an
alternative transcript preserves the original transcript. Conceivably,
the new exon could either be selected for to replace an original exon,
or to develop a specialized function as an alternative exon.
Sequence Preference for Integration
We found the chromosomal distribution of RPCs to be only slightly
skewed, with none on the Y-chromosome and the highest density on
Chromosome 15. The fact that the sequence of the Y-chromosome is only
60% complete could potentially explain the apparent lack of RPCs;
nevertheless, it is clear that there is a paucity of HMG RPCs on this
chromosome. From genomic annotations, RPCs in general are scarce on the
Y-chromosome, yet this chromosome does harbor both L1 and Alu elements,
and L1 has been shown to be expressed in testes (Ostertag et al. 2000).
The lack of RPCs on the Y-chromosome could be a phenomenon specific to
HMG genes, or a more general one. A possible explanation could be a
lack of HMG expression at a crucial stage in the male germ-line
development, causing transmission of HMG RPCs to be female-specific.
Another explanation could be the lack of transcripts only in gametes
carrying the Y-chromosome, because L1 has been shown to be active in
late-meiotic and post-meiotic male germ cells (Ostertag et al. 2000). A
similar finding was observed in the recent analysis of ribosomal
proteins' RPCs (Zhang et al. 2002), where the overall distribution of
RPCs was quite uniform, but the Y-chromosome was also underrepresented.
Because RPCs are thought to be generated by the L1 reverse
transcriptase, one might expect an increased concentration on the
X-chromosome, as L1s are twofold overrepresented on this chromosome
(Ostertag and Kazazian Jr. 2001). We do not see a similar
overrepresentation of HMG RPCs on the X-chromosome, which could mean
that the sequence being retroposed affects the target site, or that HMG
RPCs on the sex chromosomes may be selected against, as has been
suggested to explain the difference between the distribution of Alu
elements and that of L1s (Lander et al. 2001).
RPCs as Genomic Indicators
The human genome assembly and annotation projects encounter many
hurdles, some of which stem from the repetitive nature of the human
genome, as well as from the difficulty in accurately predicting genes
in genomic sequence. Some of our findings will help alleviate these
problems by providing independent information regarding potential
redundancies, segmental duplications, and gene predictions. Of 256
total entries, 48 shared 96% identities with at least one other
entry, including the RPC flanking sequence (data not shown). These
similarities could be due either to segmental duplications or assembly
flaws. Our analysis of expressed sequences yielded several matches to
mRNA models of the GenBank accession type "XM_" (data not shown).
These RPCs were typically highly similar to the HMG mRNA and still
maintained an ORF. However, no ESTs could be found that were more
similar to these RPCs than to the original HMGs, providing no real
support for their presumed expression. More likely, these were recently
created RPCs, and they were identified as model intronless genes
because they have not yet lost their ORF owing to random mutations, and
still exhibited a high degree of similarity to ESTs that were derived
from the HMG genes.
|
METHODS
|
|---|
RPCs of HMG Genes
A set of eight mRNA sequences (RefSeq were used when possible)
representing HMGA1/2, HMGB1–3, and HMGN1–3 (Fig. 1; Table 1) was
soft-masked by RepeatMasker (to mask the 6% of low-complexity
sequences present;
http://repeatmasker.genome.washington.edu/cgi-bin/RepeatMasker) and
used as a query in BLASTN searches (Altschul et al. 1990) against two
databases: the human genomic division of the nonredundant database in
GenBank (ftp://ftp.ncbi.nlm.nih.gov/genbank), and the genome build
26 (10.18.01) chromosome files
(ftp://ftp.ncbi.nlm.nih.gov/genomes/H_sapiens). These files contain
draft and finished sequence contigs that are part of the RefSeq
database (http://www.ncbi.nlm.nih.gov/LocusLink/refseq.html) and
have been assembled as explained at
http://www.ncbi.nlm.nih.gov/genome/guide/build.html.
The results were processed by adding up the local alignment endpoints
for each mRNA–genomic location pair to find the full alignment
endpoints, orientation, total match length, and overall percent
identities, while retaining the local alignment endpoints, and allowing
each genomic location only one mRNA match (generally the longest).
Alignments under 100 bp were removed from the analysis. Genomic
fragments corresponding to HMG parental genes were identified among the
genomic hits according to the following criteria: 97% identities to
mRNA, 70% mRNA length, and 4 bp overlap or gap in mRNA query
sequence between local alignments, corresponding to splice junctions.
The identified splice junctions were stored for each mRNA, as well as
the number of exons found. As a supplement to the chromosome database,
the hits to the nonredundant database were used only to identify HMG
parent genes. RPCs were defined in the chromosome files sequences as
hits in which the alignment to the mRNA spanned an identified splice
junction, as defined above. The rest of the hits were then aligned to
the mRNA using BLAST2SEQ (Tatusova and Madden 1999) with lower
stringency parameters (-W7 -G3 -E1 -q1 -FF) to find additional RPCs,
and the hits not defined as RPCs were termed PSs.
To define the RPC endpoints independently from the mRNA used for the
query, each RPC sequence with 1000 or 2000 bp from both the 5' and 3'
flanks was extracted from the databases and aligned to the previously
identified HMG parent genomic sequence with 1000 or 2000 bp of flanking
sequence on both sides, using BLAST2SEQ (Tatusova and Madden 1999). The
fetched RPC sequences were also aligned with less-stringent parameters
to the corresponding HMG mRNA. The combination of endpoints that
encompassed the longest genomic sequence was used.
Integration Sites Analysis
Chromosomal localization was determined for RPCs by finding the
physical location of the matching NT contig subsequence in the NCBI
file seq_contig.md (ftp://ftp.ncbi.nlm.nih.gov/genomes/H_sapiens),
then looking in the NCBI file ISCN800_abc for the cytogenetic location
of mapped contigs
(ftp://ftp.ncbi.nlm.nih.gov/genomes/H_sapiens/maps/mapview). The
base-pair composition was calculated for 250 bp flanking the RPC on
either side. RepeatMasker was used to find repetitive elements in these
flanking sequences, as well as for masking and subsequent use as
queries in MEGABLAST searches to look for unique hits in the
nonredundant database (Zhang et al. 2000).
Expressed RPCs
RPC sequences were soft-masked and used in BLASTN queries against
the human mRNA section of the nonredundant database and in MEGABLAST
queries against human ESTs from dbEST. Each mRNA/EST hit of 99%
identity, EST match length of 300 bp, was then compared using
BLAST2SEQ to the corresponding HMG mRNA and considered as evidence for
RPC expression only if it was 97% identical to the HMG mRNA and
therefore was not likely to be its transcript.
Expressed RPCs were aligned with the corresponding mRNA/ESTs as well as
the HMG mRNA using CLUSTAL W (Thompson et al. 1994). Alignments were
edited using Gene Doc (Nicholas et al. 1997;
http://www.psc.edu/biomed/genedoc/), which was also used to find ORFs.
Splice sites were determined near the ends of a match with an expressed
sequence by looking for the consensus GT–intron–AG in immediately
adjacent genomic sequence.
Target Site Duplications (TSDs)
For TSDs, 250 bp of sequence flanking the endpoints of RPCs were
fetched from each end. Sequences in the 3' end included the last 10 bp
of the alignment, in case the poly(A) tail was included in the mRNA.
The flanking sequences were scanned for 90% identical matches 7 bp
long. A poly(A) tract was defined as 6 As out of a moving window of
10 bp. We selected all matches found within 30 bp from both the 5'
alignment endpoint and the end of the poly(A) tract, or the 3'
alignment endpoint if no poly(A) tract was found. Of the selected
matches, we defined the two matching sequences closest to the alignment
ends as the TSD. TSDs 10 bp were aligned in two ways: the 5'-most 10
bp from each TSD and 10 bp of the sequence immediately 5' to it, or the
3'-most 10 bp from each TSD and 10 bp of the sequence immediately 3',
were aligned and analyzed using SeqLogo (Schneider and Stephens
1990; http://www.lecb.ncifcrf.gov/toms/logoprograms.html). TSDs with
ambiguities in this region were eliminated from this analysis (Fig.
7A).
|
WEB SITE REFERENCES
|
|---|
ftp://ftp.ncbi.nlm.nih.gov/genbank; human genomic division of the
nonredundant database in GenBank.
ftp://ftp.ncbi.nlm.nih.gov/genomes/H_sapiens; NCBI human genome build
26 (10.18.01) chromosome files.
ftp://ftp.ncbi.nlm.nih.gov/genomes/H_sapiens/maps/mapview; NCBI file
ISCN800_abc for the cytogenetic location of mapped contigs.
http://repeatmasker.genome.washington.edu/cgi-bin/RepeatMasker;
RepeatMasker.
http://www.informatics.jax.org/mgihome/nomen/genefamilies/
hmgfamily.shtml;
HMG chromosomal proteins nomenclature home page.
http://www.lecb.ncifcrf.gov/toms/logoprograms.html; SeqLogo.
http://www.ncbi.nlm.nih.gov/LocusLink/refseq.html; RefSeq database.
http://www.psc.edu/biomed/genedoc/; GeneDoc.
|
Acknowledgements
|
|---|
We thank Suzanne Szak and Shlomo Almashanu for many helpful
discussions; and Deanna Church, John Anderson, Wataru Fujibuchi, and
Steven Sullivan for their comments and help.
The publication costs of this article were defrayed in part by payment
of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 USC section 1734 solely to
indicate this fact.
|
Footnotes
|
|---|
3 Corresponding author. 
E-MAIL landsman{at}ncbi.nlm.nih.gov; FAX (301) 480-2288.
Article and publication are at
http://www.genome.org/cgi/doi/10.1101/gr.893803.
|
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ABSTRACT
RESULTS