Yonago Acta medica 1997;40:97–106

LL55: Selective Early Lymphoid Expression of a Murine cDNA Clone
Which Encodes a Putative RNA-Binding Protein
Takashi Baba, Saori Mitsuyoshi*, Hideya Igarashi* and Nobuo Sakaguchi*
Department of Immunology and Department of Ophthalmology, Faculty of Medicine, Tottori
University, Yonago 683 and *Department of Immunology, Kumamoto University School of
Medicine, Kumamoto 860, Japan
Specific expression of various RNA-binding protein (RNP) molecules is one of the criticalfactors that determine the development in Drosophila melanogaster and Caenorhabditis
elegans. In the present study, we found a complementary DNA clone, LL55 encoding aputative RNP. Structural comparison of nucleotide and amino acid sequences demonstrate that LL55 encoding a nuclear protein with two RNA-binding consensus motifs
and the common arginine-serine repeats, is similar to the sex-determining transformer2 (tra-2) gene of Drosophila melanogaster. The expression of 2.3 kilobase (kb) and 1.5
kb LL55 messenger RNA (mRNA) is ubiquitous in murine organs and tissues, but it isselective in the early development of fetal life. It appears as early as day 10.5 andincreases until day 15 of gestation in accordance with the expression of λ5 mRNA whichis involved in murine pre-B cell generation. Taking account of the selective expression
and the specific regulation of RNP molecules, the finding of LL55 RNP provides a new
outlook for the generation and function of the immune system.
Key words: B cell; cDNA clone (LL55); fetal development; mRNA splicing; RNP

Precursors of murine lymphocytes are generated from multipotent haematopoietic stem
cells in the fetal liver or in the adult bonemarrow (Moore and Metcalf, 1970; Huang etal.,1994). The earliest identifiable precursor
cells appear as early as day 10 in the area of theyolk sac (blood islet), and then rapidly expand
into the fetal liver by day 13 (Moore and Metcalf,
1970; Strasser et al., 1986). These progenitor
cells show surface characteristics like CD34–
Lin–Sca1+Thy1low cells, with germ line configurations of antigen receptor genes (Smith et al.,1991). Such lymphoid progenitor cells undergomolecular events for gene rearrangements ofimmunoglobulin (Ig) or T cell receptor (TCR)
loci, generating the precursor cells in the fetalliver or thymus by day 13 to day 16 (Ogawa etal., 1988; Penit and Vasseur, 1989; Rolink andMelchers, 1991). During the development oflymphoid lineage cells, a number of cell surface

molecules appear as CD117 (c-kit), CD44
(Pgp-1) and CD25 (IL-2Rα) for T lineage cells(Pearse et al., 1989; Wu et al., 1991; Godfrey
and Zlotnik, 1993; Hozumi et al., 1994) and as
CD43 and CD45 (B220) for B lineage cells(Hardy et al., 1991; Osmond et al., 1994). Although several studies have defined molecules involved in the generation of lymphoid lineage
cells by the gene targeting experiments(Kitamura and Rajewsky, 1992; Mombaerts etal., 1992; Shinkai et al., 1992; Bain et al., 1994;
Georgopoulos et al., 1994; Scott et al., 1994;
Zhuang et al., 1994), it is still not clear what themolecular mechanism that controls the early
development of lymphoid lineage cells is.
Specific expression of various RNAbinding protein (RNP) molecules is one of thecritical factors that determine the development
in Drosophila melanogaster and Caenorhabditis elegans (Parkhurst and Meneely, 1994;

Abbreviations: bp, base pair; cDNA, complementary DNA; Ig, immunoglobulin; kb, kilobase; MOPS, 3-(Nmorpholino)propanesulfonic acid; mRNA, messenger RNA; PCR, polymerase chain reaction; RACE, rapid
amplification of cDNA ends; RAG-1, recombination activating gene-1; RNP, RNA-binding protein; TCR, T cell
receptor; tra-2, transformer-2

97

T. Baba et al.

Marx, 1995; Ryner and Swain, 1995).To study
the stage specific regulation of cell development, we screened a complementary DNA(cDNA) library of a murine embryo (day 10.5)
and identified a cDNA clone named LL55 encoding an RNP that is expressed selectively during
early embryonal development. In the present
study we determined the structure and theexpression of LL55 and described the structuralsimilarity with sex-determining RNP molecule inDrosophila melanogaster (Belote and Baker etal., 1983; Amrein et al., 1988, 1990).

Palo Alto, CA) were screened by differential
screening with radioactive cDNAs frommRNAs of an adult liver, thymus, spleen, bonemarrow, WEHI-231 B cell line and BW5147 T
cell line. Positive clones with only lymphoidprobes were isolated. Nearly 800 clones wereisolated and their individual inserts were testedby the polymerase chain reaction (PCR) method
using λgt10 forward 27mer 5'(GAATTCAGC
AAGTTCAGCCTGGTTAAGT)3' and reverse
30mer 5'(TACCCTGGAAGAATACTCATAAGGAATTC)3' primers (Iwaki Glass Co, Ltd.,
Chiba, Japan). The PCR products were thenligated into T vector (Amersham Internationalplc, Bucks, United Kingdom), and the recombinant plasmid DNAs were purified to analyze
DNA sequences. In the process of these screenings and characterizations, the nucleotide
sequence data showed that a clone named LL55
with a 331 base pair (bp) insert encodes a putative RNP which is expressed selectively in
lymphoid lineage cells. Using the 331 bp probe,

Materials and Methods

Isolation of cDNA clones from fetal cDNA
library

Plasma cell

K62

BW5147

110TCpos

110TCneg

KKF

KKC

28S-

28S2.3 kb

18S-

KKB

NCKA

T cell

X63

A20

BAL17

Mature B cell

Immature B cell
WEHI-231

WEHI-279

Pre-B cell
70Z/3

18-81

40E-1

Pro-B cell

5 × 10 5 cDNA clones of a murine fetal (day10.5) cDNA library constructed in λgt10(obtained from CLONTECH Laboratories Inc.,

18S-

2.3 kb
1.5 kb

1.5 kbβ-actinβ-actinFig. 1. Northern blot analysis on various lymphoid cell lines. Total RNAs [20 µg (B cell lines) and 10 µg(Tcell lines)] extracted from various lymphoid cell lines as listed in Table 1 were separated on denaturing
agarose gel and the transferred filter was hybridized with a LL55 cDNA probe. To show the amount ofmRNA loaded, a second hybridization of the same filter was performed using human β-actin cDNA as
indicated. LL55 mRNA appears as 2.3 and 1.5 kb bands (arrows).

98Lymphoid RNA-binding protein

Table 1. Characterization of lymphoid cell lines used for Northern blot analysis
Lineage

Cell line

Developmental
stage

Ig or TCR
rearrangement

Surface expression

B cell 40E1

Pro-B

Heavy chain: DJ
Light chain: N

Ig(–)

18.81
70Z/3

Pre-B

Heavy chain: VDJLight chain: Nµ/VpreB/l5

WEHI-231
WEHI-279

Immature B

Heavy chain: VDJLight chain: VJ

IgMhigh(+)/IgDlow(+)

BAL17
A20

Mature B

Heavy chain: VDJLight chain: VJ

IgMlow(+)/IgDhigh(+)
IgG(+)/IgD(+)

X63

Plasmacyte

T cell NCKA
BW5147KKB
KKC
110TCneg

Ig(–)
α

β

CD3

CD4

CD8

Thy1

Double negative

Partially
rearranged

Not rearranged

–

–

–

+

Double negative

Rearranged

Partially
rearranged

–

–

–

+

Not rearranged Rearranged

+

+

+

+

K62
KKF
Double positive
110TCposµ/VpreB/λ5, pre-B cell receptor.

another fetal (day 11) cDNA library constructedin λgt11, kindly provided from Dr. Seiji
Miyatani, was further screened to find the fulllength clones. To determine the nucleotide sequence of the 5' side of the mRNA, the rapid
amplification of 5'-ends of cDNAs (5' RACE)
was performed according to the method described by Frohman and coworkers (1988).

filter (Amersham). The filter for fetal RNAs
and the tissue RNAs was purchased from
CLONTECH Laboratories.

Results
By differential screening of the fetal liver (day10.5) cDNA library, we isolated a cDNA clone
with a 331 bp insert that is expressed inlymphoid lineage cells. Using this LL55 probe,
Northern blot analysis on various cell lines
including pro-B, pre-B, immature B, mature B
cell lines, plasmacytoma and T cell lines demonstrated the 2.3 kilobase (kb) RNA transcript in the lymphoid cell lines (Fig. 1, Table
1). Using mRNAs from normal organs or tissues, LL55 mRNA appears as 2.3 and 1.5 kb

Northern blot analysis
Total RNAs from various lymphoid cell lines
were purified using Polytron (Kinematica AG,
Lucerne, Switzerland) with the reagent of
ISOGEN-LS (Nippon Gene, Toyama, Japan).
Northern blot analysis was performed by themethod of 3-(N-morpholino)propanesulfonic acid
(MOPS)/formaldehyde/agarose (Sambrook et al.,1989), and was transferred onto Hybond-N+99

1.5 kb50β-actinTestis1.35-

1Kidney1.5 kb2

Skeletal muscle

2.3 kb

Liver

2.4 -

3

Lung

4.4 -

4

Spleen

7.5 -

Brain

Relative LL55 mRNAs
expression

kb

Heart

Kidney2.3 kb

TestisLiver

Skeletal muscle

Spleen

Lung

Heart

Brain

T. Baba et al.

Tissues

Relative LL55 mRNAs
expression

Fig. 2. Northern blot analysis on normal organs or tissues. A: Northern blot containing approximately 2 µgof poly (A) + RNA per lane from eight different mouse tissues. It was purchased from CLONTECH
Laboratories. A second hybridization of the same filter was performed using human β-actin cDNA. B: The
expression of LL55 mRNAs was compared among normal organs or tissues by the densitometric scanning of
the Northern filter. Signals for two different sizes of LL55 mRNA were compared based on the expression of
β-actin.

2.0

LL55 (1.5 kb)
LL55 (2.3 kb)

1.5

λ5
RAG-1

1.0

0.5

0
7

11

15

17

Embryonal days
Fig. 3. Expression of LL55 mRNA during the development of mouseembryos. Northern blot containing approximately 2 µg of poly (A)+RNA per lane from mouse embryos of different developmental stages asdays 7, 11, 15 and 17. It was purchased from CLONTECH Laboratories. The filter was stripped of radioactivity and hybridized with aradioactively labeled [α-32P]dCTP mouse λ5, mouse RAG-1 and human
β-actin cDNA control probe. Relative amounts of each λ5, RAG-1 and
LL55 specific mRNA species were estimated by densitometric analysis.
Signal intensities were normalized on the basis of the β-actin signals foreach sample.

100

bands (Figs. 2A and B). LL55
is expressed in the heart,spleen, lung, liver, kidney andthe testis, but the expression is
weak in the skeletal muscle
and the brain. The expressionof LL55 mRNA was mostabundant in the testis by ourNorthern blot analysis. The
relative expression of two
forms of LL55 mRNA wascompared based on the expression of β-actin (Fig. 2B). LL55
mRNA from the heart, liver andthe kidney is predominantly 2.3
kb, while the other organs
express similar amounts ofmRNA with both sizes.
The expression of LL55
mRNA was studied during thedevelopment of lymphoidorgans in the fetal life. The

Lymphoid RNA-binding proteinASR-rich RNP-2 RNP-1 G-rich SR-richpoly A
1

ATG
2

STOP
4

3

5

6
ORIGINAL
5'-RACEclone-9 (λgt10)
poly A (17A's)

clone-14

poly A (28A's)

clone-22
clone-4
clone-8clone-17
clone-18

B

clone-6

clone-20
INVERTED

Fig. 4. Relative map of the LL55 clones. A: Relative map of the clones isolated using the original (331 bp)
cDNA probe. Only clone 5'-RACE was isolated by the 5'-RACE method. Narrow open boxes indicate theconsensus sequence of clone-17. Wide open boxes indicate the coding region of LL55 polypeptide (288amino acids) with RNP-1 domain ( ), RNP-2 domain ( ), SR-rich domain ( ) and G-rich domain ( ).
The longest clone clone-17 contains the 4 kb insert. Clone-14 and clone-22 have polyadenylation signal andpoly A. B: Clone-6 and clone-20 contain inversions of their sequences (open arrow box). Clone-20 contains
an inversion of the RNP-1 domain.

relative expression of the transcripts is shown
based on the expression level with β-actin
probe for the control of RNAs. LL55 mRNAappears with the sizes of 2.3 kb ( ) and 1.5 kb( ) at day 11 and is up-regulated during fetal
development, reaching the maximum at day 15(Fig. 3). Markers for lymphoid generation werealso monitored on the same blot as RAG(recombination activating gene)-1 ( ) for thegene rearrangement of the antigen receptor
genes (Oettinger et al., 1990), and λ5 ( ) for
pre-B cell generation (Sakaguchi and Melchers,1986). LL55 expression is up-regulated inparallel with the expression of λ5 mRNA.

Using the LL55 cDNA probe, the longer
cDNA clones were isolated and their sequenceswere determined respectively. Figure 4A
shows the relative map of the clones. One of theclones named clone-17 spans for a possible
longest reading frame. To detect the alternative
splicing of LL55 RNA, the PCR method withseveral combinations of primers was performed
for the analysis of cDNA clones isolated in theschematic orientation (Fig. 4A). By Northernblot analysis, the most abundant class of mRNAis 2.3 kb RNA with all four kinds of domains
such as 5' SR, RNP-2, RNP-1, G-rich and 3' SR.
The next class contains G-rich and 3' SR

101

T. Baba et al.

-143

gaattcCGGGGTGCGGAGCGTGTCGGCTTCGAGCTCCTCGCAAAAGTGTGGCGTCGCGC-85-84 TAGGCTGCGGCGGAGCCTCTGTAAGGAAGGCGGGAGCTTGACAGCTTCAGGAAAGGCCCG-25(sequence data not shown).
By screening a fetal (day-24 TAGCGGTGACAGCGACCCGGAGTCATGAGCGACAGCGGCGAGCAGAACTACGGCGAGCGG
36
M S D S G E Q N Y G E R
1211.5) cDNA library with the+1
LL55 probe containing RNP37 GAATCCCGTTCTGCTTCCCGAAGTGGAAGTGCTCACGGATCGGGGAAATCTGCACGGCAT
96
1 domain, we obtained 33
13 E S R S A S R S G S A H G S G K S A R H
32
clones with different sizes of
97 ACACCTGCAAGGTCTCGCTCCAAGGAAGACTCAAGGCGTTCTAGATCAAAGTCCAGGTCC
156
insert DNAs. Analysis of
33 T P A R S R S K E D S R R S R S K S R S
52
two clones with 4.2 kb inserts
157 AGGTCTGAATCTAGGTCTAGATCCAGAAGAAGTTCTAGAAGGCATTATACAAGGTCACGA
216
53 R S E S R S R S R R S S R R H Y T R S R
72
demonstrated that bothclones contain an identical
217 TCACGATCTCGCTCGCATAGACGATCCCGGAGCAGGTCTTACAGCCGAGATTATCGCAGG
276
73 S R S R S H R R S R S R S Y S R D Y R R
92
sequence but several clones
show the inversion of the
277 CGCCACAGCCACAGCCATTCTCCCATGTCTACTCGAAGGCGTCATGTTGGGAACCGGGCA
336
93 R H S H S H S P M S T R R R H V G N R A
112
RNP-1 domain, creating a
truncation of protein coding
337 AATCCTGACCCCAACTGTTGTCTTGGCGTGTTTGGGTTGAGCTTATACACCACAGAAAGA
396
113 N P D P N C C L G V F G L S L Y T T E R
132(Fig. 4B; clone-6 and cloneRNP-2
397 GACCTAAGAGAAGTGTTCTCTAAATATGGCCCCATTGCTGATGTGTCTATTGTATATGAC
45620) (data not shown). These133 D L R E V F S K Y G P I A D V S I V Y D
152
results suggest that the ex457 CAGCAATCTAGACGTTCAAGAGGATTTGCCTTTGTATATTTTGAAAATGTAGACGATGCC
516
pression of the LL55 protein
153 Q Q S R R S R G F A F V Y F E N V D D A
172
might be also controlled
RNP-1
517 AAGGAAGCTAAAGAACGTGCCAATGGAATGGAGCTTGATGGGCGTCGAATTAGAGTCGAT
576
under alternative RNA splic173 K E A K E R A N G M E L D G R R I R V D
192
ing.
577 TTCTCTATAACAAAAAGGCCCCATACCCCAACACCAGGAATTTATATGGGGAGACCCACT
636
Figure 5 shows the nucle193 F S I T K R P H T P T P G I Y M G R P T
212
otide and the amino acid se637 TATGGCAGTTCTCGCCGCCGAGACTATTATGACAGAGGGTACGATCGGGGTTATGATGAC
696
quences of LL55. There is a
213 Y G S S R R R D Y Y D R G Y D R G Y D D
232poly-A sequence on one end
697 CGGGACTATTACAGCAGATCATACAGAGGAGGAGGTGGTGGAGGAGGTGGATGGAGAGCA
756
with a polyadenylation signal
233 R D Y Y S R S Y R G G G G G G G G W R A
252
AATAAA located 28 bp up757 GCTCAAGACAGGGATCAGATTTACAGAAGACGGTCACCTTCTCCTTACTACAGTCGTGGA
816
stream, determining the ori253 A Q D R D Q I Y R R R S P S P Y Y S R G
272
entation of the cDNA clone.
817 GGATACAGGTCACGTTCTCGATCACGATCCTACTCACCTCGTCGCTACTAAAGCATGAAG
876
273 G Y R S R S R S R S Y S P R R Y *288
The open reading frame encodes a putative protein with
877 TTGAAGACTTTCTGAAACCTGCCATAGAGCTGGGATATTGTTTGTGGACAATATTTTCTA
936
937 TTGTCTCCTGTTTAAAAAGTGAACAGTGCCTAGTGAAGTTAGGTGACTTTTACACCTTTT
996
288 amino acids starting from
997 ATGATGACTACTTTTGGTGGAGTTGAAATGCTGTTTTCATTCTGCATTTGTGTAGTTCGG
1056
1057 TGCTTTGTTGCAAAGTTAAGTGTTTTCAGAAAAGTATGTTTTGCATGTATTTTTTTACAG
1116
the methionine at the nucleo1117 TCCAAATTTTGACTGCTGAGAAGTTTCTATTGTAAAAAACTTCATTTAAAAGGTTTTTCT
1176
tide number 143 and ending
1177 ACTGAATCCAGGGTATTCTGAAGATCGAAGCCTGTGTGTAAAATGCTACCAGATGGCAAA
1236
1237 AAGCAACAATAAACAAGTTTGATTTTTACTTTTCTTTCTAAATATCAATGCTTAACCAGA
1296
as TAA at the nucleotide num1297 ACCATTCTTAAGTTATCAGTAAAAAAGTAAAAAGTTAAATAAATGTCTTTCAGGAGTCCA
1356
1357 TGAAACTTGCCATATTCAATATACCTGCGATTAAGTGTTAAAAAATACGCTGTAACTCTG
1416
ber 865. The first methionine
1417 TACTGCTAGTATTAGAACAAAACTCTCCCCATACAGCAAATGCTTAATGCTTACATTAAT
1476
codon does not match the1477 GTGGATGTGTCGGCCTTTATGTAATCTGTATTATATATAGCAGGGAATAAGAGCTACAGA
1536
1537 TACATGCCTTTAAAGGGCTGTTTCTTAAGGCTGTTACAAGGAGATAATGGTATTTCAACT
1596
consensus sequence for opti1597 AATTATCAGCAAGTGACAATACATTCCACCACAGATGGACTCTTGTTCTTCTAGCGTTTA
1656
1657 GACTATATGAAAATACTGGGTGCTTCAAAGTATGGAAGGAGCGAAGGGATCATCAAACCT
1716
mal initiation (Kozak, 1987).
1717 TGTCATGGATGAAGACTGCCTGTTCATTTTTTAAATATGATTTTCAGGTCCTTTGCTTAC
1776
1777 CAAAGGAGGCCCAATTCACTCAAATGTTTTGAGAACTGTTTAAATAAAGGCAAGAAAAAT1836
The nucleotide sequence of1837 TGCTGATGCAAAAAAAAAAAAAAAAAAAAAAAAAAAA
1873
this clone showed the hoFig. 5. Nucleotide and amino acid sequence of LL55. Homologous mology to a known RNP ofstretches for RNP is indicated by an open box for RNP-1 and RNP-2. The Drosophila, the sex determinpoly adenylation signal AATAAA is indicated by an underline.
ing protein of transformer-2(tra-2) (49.2% identity indomain, but lacks 5' SR, RNP-2 and RNP-1 nucleotide sequence and 38.2% in amino acid(data not shown). These two forms are also sequence) (Belote and Baker, 1983; Amrein etconfirmed with cDNA clones isolated using 5' al., 1988, 1990). And a part of the nucleotide
RACE method (Fig. 4A; clone 5'-RACE) sequence of this clone corresponds to the SIG41
102

Lymphoid RNA-binding protein

A
RNP-2
hexamer

RNP-1
octamerFly Sxl #1

126 NLIVNYLPQDMTDRELYALFRAIGPINTCRIMRDYKTGYSFGYAFVDFTSEMDSQRAIKVLNGITVRNKRLKVSYARPGG

205

Fly Sxl #2

212 NLYVTNLPRTITDDQLDTIFGKYGSIVQKNILRDKLTGRPRGVAFVRYNKREEAQEAISALNNVIPEGGSQPLSVRLAEE

291

Fly Tra-2

100 GVFGLNTN--TSQHKVRELFNKYGPIERIQMVIDAQTQRSRGFCFIYFEKLSDARAAKDSCSGIEVDGRRIRVDFSITQR

177

|||||
Mouse LL55||| | |||||| | || ||| | |||||||| | |||||||||||| |121 GVFGLSLY--TTERDLREVFSKYGPIADVSIVYDQQSRRSRGFAFVYFENVDDAKEAKERANGMELDGRRIRVDFSITKR

199

B
Fly Tra73 SRSRSQSSIRESRHRRHRQRSRSRNRSRSRSSERKRRQRSRSRSSERRRRQRSPH
127
Fly Tra-2 #1
39 RSDYDYCGSRRHQRSSSRRRSRSRSSSESPPPEPRHRSGRSSRDRERMHKSREHP
93
RGKAPRSFSPRRGRRVYHDRSASPYDNYRDRYDYRNDRYDRNLRRSPSRNRYTRNRSYSRSRSPQLRRTSSRY 264
Fly Tra-2 #2
192
Mouse LL55 #1 14 SRSASRSGSAHGSGKSARHTPARSRSKEDSRRSRSKSRSRSESRSRSRRSSRRHYTRSRSRSRSHRRSRSRSYSRDY 90
WRAAQDRDQIYRRRSPSPYYSRGGYRSRSRSRSYSPRRY 263
Mouse LL55 #2 192

Fig. 6. A: Alignment of LL55 protein RNP. Conserved features among RNP domains of LL55 and otherproteins. Regions of sequence similarity among several SR-proteins and LL55 are arranged for the bestalignment of conserved residues within sub-domains of RNA binding motif. The RNP-1 and RNP-2 elements
are shown in reverse type, and eight other highly conserved positions are show in narrow open boxes. The
numbers on the left and right ends of the sequence indicate the positions of each SR proteins in the respective
amino acid sequences. B: Alignment of SR-rich domain of LL55 protein. Comparison of SR domains of
LL55 to SR domains of SR-protein family. Amino acid identities and semiconservative changes are indicatedby shading.

gene that is induced by silica treatment inmurine macrophage cells and is homologous to
the RA301 gene that is induced by hypoxia/
reoxygenation in rat astrocytes (Matsuo et al.,
1995; Segade et al.,1995). The putative LL55
protein contains apparent consensus motifs for
the RNA-binding protein with highly conserved
oligopeptide cores called RNP-1 (octapeptide)and RNP-2 (hexapeptide), both of which aresurrounded by the serine-arginine repeats (SRMouse SIG41
LL55
Rat
RA301
Fly

Tra

Fly

Tra-2

Human X16

197aadomain) spanning to 100 amino acids in theamino terminal and to 50 amino acids in the
carboxyl terminal regions (Figs. 6A and B).
This protein also has a glycine-rich domain including 8 glycine residues. It may act as a hinge
region between N-terminal and C-terminal
domains. The proteins with RNP and SR
domains are classified as the SR-protein family,
whose function is essential for RNA splicing.
Compared with a number of SR proteins in
Drosophila melanogaster,mouse, rat and human, LL55
288aa
contains two RNA-binding
domains in the middle of theprotein and SR domains inboth amino- and carboxylterminals (Fig. 7)
264aa
RNP1
RNP2
SR-richG-rich164aa

Fig. 7. Diagram of the SR family proteins. Overall structure of LL55 polypeptide and several RNP proteinsare compared based on the key amino acids commonly seen among the members of SR-protein family.
Various domains represented include RNA binding motif with internal octapeptide RNP-1 core (light shadedbox), hexapeptide RNP-2 core (dark shaded box), SR-rich domain (diagonally striped box) and glycine-richdomain (black box).

103

T. Baba et al.

Discussion
In the present study, we isolated a cDNA clone
expressed in fetal development which potentially encodes an RNP with RNA splicing
activity. The putative protein of LL55 has
RNP- and SR-domains and classified as an SR
protein family. In the initial screening, it was
expressed specifically in the lymphoid lineage
cells. By using the specific probe which does
not cross-react with other members of the SR
family, LL55 is expressed ubiquitously in manykinds of organs and tissues as shown in Figs. 2Aand B, but is preferential in the lymphoidorgans and the testis. Interestingly, LL55
mRNA appears in fetal development as early as
day 11 of gestation when lymphoid generation
has just occurred (Fig. 3), especially in the fetalliver detected by the in situ RNA hybridization
method (data not shown). This expression
profile suggests that LL55 RNP might play
critical role in the generation of early lymphoid
precursor cells.
Alternative splicing is one of the main
mechanisms including other gene duplications
and gene rearrangements that evolve to produce
protein diversity at the level of the primarystructure. Several reports concerning the tissue
specific expression of human or murine RNPs
provide information about physiological
function in the development and activation of
eukaryotic cells (Vellard et al., 1992; Diamond
et al., 1993). The gene rearrangement of Ig and
TCR genes is an efficient mechanism for thegeneration of protein diversity from a limited
number of genes. It irreversibly changes thegenetic content of the cells, committing thecells to a process of terminal differentiation.
Alternative splicing offers many advantages. It
does not change the genetic content of the cellnor is it irreversible. Splicing pathways neednot be discarded to use one of the molecules,
creating a stable diversity during early developmental stages.
In the immune system, however, it remains
to elucidate the expression and the function oflymphoid specific RNPs regulating RNA splic-ing of a variety of lymphoid specific molecules
during development. Previously, Ayane andcoworkers (1991) reported a gene named X16
encoding a protein with a nucleotide binding
motif, which is expressed differentially inhuman pre-B cells (Fig. 7). Expression of LL55
mRNA is more restricted to the earlier stages ofthe lymphoid development than X16 gene.
Lymphoid RNPs could play roles at various
phases in the development of the immune
system. Expression of Ig and TCR genes
probably requires a specific RNA splicing
process allowing the expression of a functional
JH sequence without the co-expression of 3'-JH
sequences (Hiramatsu et al., 1995). Recently,
Aoufouchi and coworkers (1996) demonstrated
that the intronic sequence influences κ geneRNA splicing in a cell-free system, suggesting
the important role of RNA splicing in theregulation of Ig expression during thedevelopment of B lineage cells. Differential
RNA splicing events in the lymphoid system
are reported: the activation of antigen receptor
positive B cells switches the pattern of RNA
splicing from a membrane form to a secreted
form in Ig mRNAs (Early et al., 1980); lymphoid differentiation accompanies lineage
specific RNA splicing of CD45 as CD45RO for
B cells and CD45RA, RB, RC for T cells(Screaton et al., 1995). Alternative RNA
splicing and production of different mRNAs
leads to co-expression of µ and δ or membraneand secreted immunoglobulin, and coexpression of multiple heavy chain clones inindividual B cells. Alternative RNA splicing
plays an important role in the immune system.
From Northern blot analysis LL55 mRNAappears in sizes 2.3 and 1.5 kb during the embryonal stage, although the transcript of the
LL55 gene varies during the development. A
number of LL55 clones were isolated from thefetal cDNA library and their sizes were studied
by restriction mapping and PCR analysis using
several combinations of LL55 primers. One of
the clones showed the apparent inversion of the
RNP-1 domain (Fig. 4B; clone-20), which
probably caused the defective function in RNA
splicing activity. It has been often shown thatmany SR proteins show regulatory function by104

Lymphoid RNA-binding protein

alternative splicing of their own RNA
molecules to create a truncated form of mRNA
(Bingham et al., 1988). Although our results
did not clearly demonstrate the function of the
SR protein in the LL55 molecule for thedevelopment of lymphoid cells, the alternative
form of mRNA was shown by Northern blot
analysis and the RT-PCR method. Especially,
the inverted form of LL55 would be a sign for
the RNA splicing function of the LL55 molecule. We tried to determine a protein molecule
encoded by LL55 using the in vitro transcription and translation method. The protein
molecule synthesized from the LL55 cDNA
clone introduced into pBluescript vector
showed an apparent molecular weight of 35
kDa, 30 kDa and 19 kDa on SDS-PAGE
analysis (data for the paper prepared by Dr.
Hideya Igarashi and others). It will benecessary in the future to study the RNA
splicing function of the RNA molecules used in
the differentiation and activation of lymphoidcells.

Acknowledgments: We wish to thank Dr. Seiji
Miyatani (Research Institute for Biosciences,
Science University of Tokyo) for his kind advice andcooperation in this study, and Prof. Akihiko Tamai(Department of Ophthalmology, Faculity of
Medicine, Tottori University) for his usefulsuggestion and critical reading of the manuscript.
This work was supported by grants from the
Ministry of Education, Science, Culture and Sportsof Japan and a grant from the Japan Science Promotion Agency.

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(Received April 21, Accepted May 12, 1997)

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