Vol.3, No.4B, 36-48 (2013) Open Journal of Animal Sciences
http://dx.doi.org/10.4236/ojas.2013.34A2005
A phylogenetic study of Drosophila splicing
assembly chaperone RNP-4F associated
U4-/U6-snRNA secondary structure
Jack C. Vaughn*, Sushmita Ghosh, Jing Chen
Department of Biology, Cell Molecular and Structural Biology Program, Miami University, Oxford, USA;
*Corresponding Author:
Received 15 August 2013; revised 23 September 2013; accepted 1 October 2013
Copyright © 2013 Jack C. Vaughn et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The rnp-4f gene in Drosophila melanogaster
encodes nuclear protein RNP-4F. This encoded
protein is represented by homologs in other
eukaryotic species, where it has been shown to
function as an intron splicing assembly factor.
Here, RNP-4F is believed to initially bind to a
recognition sequence on U6-snRNA, serving as
a chaperone to facilitate its association with
U4-snRNA by intermolecular hydrogen bonding.
RNA conformations are a key factor in spli-
ceosome function, so that elucidation of chang-
in g seconda ry structures for interacti ng sn RNAs
is a subject of considerable interest and impor-
tance. Among the five snRNAs which participate
in removal of spliceosomal introns, there is a
growing consensus that U6-snRNA is the most
structurally dynamic and may constitute the
catalytic core. Previous studies by others have
generated potential secondary structures for
free U4- and U6-snRNAs, including the Y-shaped
U4-/U6-snRNA model. These models were based
on study of RNAs from relatively few species,
and the popular Y-shaped model remains to be
systematically re-examined with reference to the
many new sequences generated by recent ge-
nomic sequencing projects. We have utilized a
comparative phylogenetic approach on 60 di-
verse eukaryotic species, which resulted in a
revised and improved U4-/U6-snRNA secondary
structure. This general model is supported by
observation of abundant compensatory base
mutations in every stem, and incorporates more
of the nucleotides into base-paired associations
than in previous models, thus being more en-
ergetically stable. We have extensively sampled
the eukaryotic phylogenetic tree to its deepest
roots, but did not find genes potentially encod-
ing either U4- or U6-snRNA in the Giardia and
Trichomonas data-bases. Our results support
the hypothesis that nuclear introns in these
most deeply rooted eukaryotes may represent
evolutionary intermediates, sharing characteris-
tics of both group II and spliceosomal introns.
An unexpected result of this study was discov-
ery of a potential competitive binding site for
Drosophila splicing assembly factor RNP-4F to a
5’-UTR regulatory region within its own pre-
mRNA, which may play a role in negative feed-
back control.
Keywords: RNP-4F; snRNA Secondary Structure;
U4-/U6-snRNA Phylogeny; Spliceosome Evolution
1. INTRODUCTION
Drosophila melanogaster, which is a cosmopolitan
holometabolous insect found in all warm environments,
has been an important model organism for genetic, mo-
lecular, cellular and physiological studies for over a cen-
tury. Its small size (usually 2 - 4 mm), short life cycle (10
- 14 days at 25˚C), high reproductive rate (an adult fe-
male can lay 400 - 500 eggs in 10 days), completely se-
quenced and largely annotated genome, well-developed
techniques, and evolutionarily-conserved molecular path-
ways all contribute to making Drosophila a research
paradigm. It has been predicted that about 75% of human
disease genes have clear homologs in D. melanogaster
[1,2], an observation leading to the extensive use of
Drosophila which has led to advances in the improve-
ment of human health.
The long-term objective of our research is to under-
stand evolutionarily-conserved cellular, developmental,
molecular and genetic mechanisms behind regulation of

J. C. Vaughn et al. / Open Journal of Animal Sciences 3 (2013) 36-48 37
genes which encode intron splicing assembly factor pro-
teins, a topic about which relatively little is known. The
system which we are currently using to address these
questions is the Drosophila rnp-4f gene, which encodes
splicing assembly factor RNP-4F, and we are concen-
trating on mechanisms of posttranscriptional level regu-
lation [3-11]. This protein is believed to play a direct role
during spliceosome assembly by acting as a chaperone to
unwind U6-snRNA and thus facilitate its association with
U4-snRNA via intermolecular h ydrogen bonding [12-1 6].
In the course of our work, we became interested in sec-
ondary structure interactions within the Drosophila
U4-/U6-s nRNA duplex.
The major or U2-type molecular pathway for removal
of spliceosomal in trons has been extensively studied [re-
viewed in 17, 18], and shown to require direct participa-
tion of five trans-acting small nuclear uracil-rich RNAs
(snRNAs) termed U1, U2, U4, U5 and U6. These RNAs
are each associated with specific sets of proteins to yield
the corresponding biologically active snRNPs, which
progressively interact with pre-mRNAs and with each
other during the ensuing spliceosomal assembly. In addi-
tion to these snRNAs, about 70 different snRNP proteins
and more than 100 non- snRNP proteins have been shown
to be spliceosomal components [reviewed in 19]. For
example, the essential Saccharomyces cerevisiae pre-
mRNA splicing protein Prp24, rep resented in Drosophila
by its ortholog RNP-4F and in human by p110 [13,14]
facilitates U4- and U6-snRNA pairing during spliceoso-
mal assembly [16].
A succession of snRNA conformational changes ac-
companies steps in the splicing pathway, which are es-
sential in generation and function of the catalytic struc-
ture. Elucidation of the changing secondary structures of
the interacting snRNA molecules is therefore a subject of
considerable interest and importance. The comparative
phylogenetic approach [20,21] generates models in
which existence of potential biologically significant
stem-loops can be established by observation of com-
pensatory base mutations in diverse species, and has
proven to be a powerful technique. The original Y-shaped
U4-/U6-snRNA duplex secondary structure model [12]
was based on this methodology by comparing yeast,
fruit-fly, plant and human sequences. Subsequent studies
have shown that RNAs from various species can also be
folded in accordance with this model [22-26]. However,
no attempt has ever been made to systematically re-ex-
amine the original model itself, utilizing the relative
abundance of new sequences now available for analysis.
2. MATERIALS AND METHODS
2.1. Selection of U4- and U6-snRNA
Sequences
We began by utilizing the original Small RNA Data-
base [27] as a source for sequences published early. We
then carried out GenBank searches, followed by BLAST
searches (http://www.ncbi.nlm.nih.gov/BLAST) in which
bait sequences were derived from the major phylogenetic
levels. Finally, the number of sequences available for
study was further increased from early published work
not submitted to GenBank. The BLAST search was more
successful in finding U6-snRNAs, owing to their ex-
tremely high sequence conservation. We did not use
every sequence found, excluding for example those from
eleven other Drosophila species [28] and also different
species of Saccharomyces, since their inclusion would
add little additional understand ing due to having v irtually
identical sequences within a genus. This exercise (Table
1) yielded 42 U4- and 56 U6-snRNAs, of which 38 were
both available in a given species and deemed optimal for
our study. In total, sequences from some 60 different
species were included in our study.
2.2. Alignment of U4- and U6-snRNA
Sequences
All sequences selected for this study were individually
aligned with reference to the corresponding Drosophila
genes using the ClustalW program (http://align.genome.jp),
and the resulting alignment was further refined by eye.
Finally, the alignment was adjusted using the emerging
secondary structure results, to assure that homologous
nucleotides would be compared for evidences of com-
pensatory base mutations. The final alignments (not
shown) included as few deletions (gaps) and insertion s as
possible, while generating the maximum number of
matchi n g re sidues.
2.3. Strategy for U4-/U6-snRNA Duplex
Secondary Structure Determination
We elected to start completely from the beginning in
deriving our secondary structure model, in contrast to
merely modifying existing models, to optimize the
chances of identifying structural components not previ-
ously recognized. We began by utilizing version 3.6 of
the Mfold program (http://mfold.rna.albany.edu) [29] for
the two genes individually from Drosophila melanogaster
(fruit-fly), Homo sapiens (human), Arabidopsis thaliana
(plant), Kluyveromyces lactis (yeast) and Trypanosoma
brucei (flagellate). GenBank accession numbers are
given in Table 1 . These structures contained a variety of
potential stem-loops, and were combined to include only
stem-loops held in common. The resulting U4- and
U6-snRNA structures were then combined to accommo-
date base-pairing between the two molecules in the two
closely adjacent U6 locations previously determined by
photochemical cross-linking in mammalian snRNAs [30]
nd by subsequent observation of compensatory base a

J. C. Vaughn et al. / Open Journal of Animal Sciences 3 (2013) 36-48
38
Table l. U4 and U6 RNA sequences utilized in this study.
Organism GenBank Accession Number or Reference
U6-snRNA U4-snRNA
Animalia, Vertebrate
Homo sapiens (human) X07425 X59361
Pan troglodytes (c h impanzee) AC146131 NW_001223167
Macaca mulatta (monkey) NW_001218112 NW_001096649
Mus musculus (mouse) X06980 AC159539
Rattus norvegicus (rat) AC120800 K00477
Canis familiaris (dog) AC188530 NW_876282
Bos taurus (cattle) NW_001492849 NW_001493540
Sus scrofa (pig) CR956385 -----
Equus caballus (horse) NW_001799704 NW_001799734
Monodelphis domestica (opossum) NW_001581906 NW_001584232
Ornithorhynchus anatinus NW_001794177 NW_001765942
(duck-billed platypus)
Gallus gallus (chicken) NW_001471627 M14136
Xenopus tropicalis (frog) M31687 -----
Danio rerio (zebrafish) CU466287 NW_001514552
Animalia, Invertebrate
Drosophila melanogaster (fruit-fly) X06669 D00043
Aedes aegypti (mosquito) AAGE02013372 -----
Anopheles gambiae (mosquito) NZ_AAAB02008807 -----
Culex pipiens (mosquito) AAWU01008690 AAWU01009244
Apis mellifera (honey bee) NW_001253045 -----
Nasonia vitripennis (jewel wasp) NW_001815737 AAZX01001234
Bombyx mori (silkworm moth) AADK01011346 DQ861919
Tribolium castaneum (flour beetle) AC154132 NW_001092869
Tachypleus tridentatus X53789 -----
(horseshoe crab)
Ascaris lumbricoides (nematode) L22252 L22250
Caenorhabditis elegans (nematode) X07829 X07828
Schistosoma mansoni (trematode) L25920 -----
Taenia solium (tapeworm) AF529186 -----
Ly t echinus variegatus (sea urchin) ----- U37266
Strongylocentrotus purpuratus X76389 NW_001323459
(sea urchin)
Fungi, Ascomycota
Saccharomyces cerevisiae X12565 Siliciano et al. (1987)
(budding yeast)
Schizosaccharomyces pombe X14196 X15491
(fission yeast)
Kluyveromyces lactis NC_006042 Guthrie & Patterson (1988)
Candida albicans EU144231 EU144229
Va nde rwaltoz yma po lyspor a NZ_AAZN01000268 -----

J. C. Vaughn et al. / Open Journal of Animal Sciences 3 (2013) 36-48 39
Continued
Ashbya gossypii NC_005788 -----
Fungi, Basidiomycota
Erythrobasidium hasegawianum Tani & Ohshima (1991) D63682
Puccinia graminis AAWC01000866 -----
Coprinopsis cinerea AACS01000244 -----
Phanerochaete chrysosporium AADS01000210 -----
Amoebozoa, Mycetozoa
Dictyostelium discoideum AY953942 AY918063
(sl ime mold )
Physarum polycephalum ----- X13840
(sl ime mold )
Amoebozoa, Con osa
Entamoeba histolytica U43841 BK006131
Viridiplantae, Eudicot
Arabidopsis thaliana (thale cress) X52527 X67145
Vicia faba (broad bean) Solymosy & Pollak (1993) Solymosy & Pollak (19 93 )
Pisum sativum (pea) Solymosy & Pollak (1993) X15933
Solanum lycopersicum (toma to) X51447 -----
Solanum tuberosum (potato) S83742 -----
Populus trichocar pa (Poplar) NC_008469 NC_008470
Viridiplantae, Monocot
Oryza sativa (rice) NC_008405 DQ649301
Triticum aestivum (wheat) X63066 -----
Zea mays (maize) ----- Solymosy & Pollak (19 93 )
V iridiplantae, Algae
Chlamydomonas reinhardtii X71486 X71485
Alveolata, Cilliophora
Tetrahymena thermophila Orum et al. (1991) Orum et al. (1991)
Alveolata, A picomplexa
Plasmodium falciparum EF419774 EF140769
Euglenozoa
Trypanosoma brucei (flagellate) X57 04 6 Solymosy & Pollak (1993)
Crithidia fasciculata (flagellate) X78550 AF326336
Leishmania tarentolae (flagellate) ----- X97621
Leishmania mexicaca (flagellate) X82228 -----
Leptomonas seymouri (flagellate) X78552 AJ245951
Phytomonas sp. (flagellate) X82229 -----
mutations [12], which resulted in further simplification
of potential stem-loops in the predicted duplex RNA
structure. Compensatory base changes were then entered
onto the Drosophila duplex structure in comp arison with
the five species originally used to begin the study
(above), using the alignment to assure that homologous
nucleotides were being compared. We adopted the crite-
rion [20] that existen ce of a helix is considered proven if
there are at least two base-pair replacements. Stems as
short as two base-pairs are acceptable if compensatory
base changes can be demonstrated (Carl Woese, personal
communication). Finally, the provisional model was
compared to every species utilized in the study (Table 1),
to determine the extent to which the resulting structure
was univ ersal. When an otherwise proven ste m-loop was
found to be absent from any taxonomic level, the timing

J. C. Vaughn et al. / Open Journal of Animal Sciences 3 (2013) 36-48
40
of that loss was charted with reference to the eukaryotic
phylogenetic tree [31].
3. RESULTS AND DISCUSSION
3.1. An Improved General Secondary
Structure Model for U4-/U6-snRNA
The derived U4-/U6-snRNA duplex secondary struc-
ture model is shown in Figure 1, and structures from
representative species at different taxonomic levels in
Figures 2(a)-(h). A relatively large proportion of all nu-
cleotides are base-paired in our U4-/U6-snRNA model.
For example, in Drosophila 58% are base-paired in U4
and 63% in U6, whereas in the Y-shaped model the cor-
responding numbers are 58% and 33%. Four stem-loops
(I-IV) are found to be present in the U4 structure for
most species, so that our model both confirms and ex-
tends the secondary structure for free U4-snRNA previ-
ously proposed [32] using the phylogenetic approach
with far fewer species. The existence of stem-loop IV in
free U4-snRNA, proposed by the same authors, is also
confirmed for all species studied by us. The overall con-
formation of the structure shown in our model is very
similar in every species examined, with the exception of
stem-loop III in U4-snRNA which is further discussed in
Section 3.2. Each stem in our model has been proven by
Figure 1. General secondary structure model for Drosophila U4-/U6-snRNA duplex. The two RNAs inter-
act by base-pairing within regions designated DS I and DS II. Compensatory base changes which prove the
structure illustrated are boxed and were identified in the alignment with reference to the structures derived
for H. sapiens, A. thaliana, K. lactis and T. brucei. The range of stem lengths found between different spe-
cies in our study is shown beside each stem. Stem-loop IV in free U4-snRNA (large box) is disrupted upon
binding to U6-snRNA. The putative SM-binding site (SM) is indicated. An RNA recognition motif (RRM)
in chaperone RNP-4F/Prp24/p110 binds primarily to a tract within free U6-snRNA nucleotides #38-57 (13),
which is indicated by a heavy vertical overlay.

J. C. Vaughn et al. / Open Journal of Animal Sciences 3 (2013) 36-48 41
observation of numerous compensatory base mutations.
Species within the flagellate group Euglenozoa were
found to have the shortest overall U4- and U6-snRNA
lengths (compare D. melanogaster in Figure 1 with T.
brucei in Figure 2(h)). Despite the close similarity in
conformation among species, nearly all stem lengths are
however quite variable (Figure 1). The most consistent
stem length is in U4 stem I, which ranges from 10 - 13
base pairs and is always interrupted by a structurally
conserved bulge loop. A conspicuous highly conserved
sequence tract in U4 is the putative SM-binding site,
located near the 3’-end between stem-loops II and III,
which matches the consensus sequence AU [4-6] G. Our
study confirms the universality of the two major inter-
molecular base-paired zones of contact between the two
RNA molecules (DS I and DS II) as originally proposed
[12], with many examples of compensatory base muta-
tions.

J. C. Vaughn et al. / Open Journal of Animal Sciences 3 (2013) 36-48
42
Figure 2. Representative U4-/U6-snRNA secondary structures from phylogenetically diverse species, folded according to our
general model. (a) H. sapiens; (b) S. cerevisiae; (c) T. thermophila; (d) P. falciparum; (e) D. discoideum; (f) A. thaliana; (g) C.
reinhardtii; (h) T. brucei. Labeling is as in Figure 1.
The U6-snRNA nucleotide sequence is relatively
highly conserved, in comparison with that for U4. Three
stem-loops are also present in the U6 structure in our
duplex model, which is in contrast to the Y-shaped model
in which only stem-loop I is shown. In our model the
3’-end of U6-snRNA is incorporated into the structu re to
form stem-loop II in every species examined, albeit in
some cases with a central bulge loop or absence of
base-pairing at the top of the stem. U6 stem-loop II is
proven by observation of compensatory base mutations,
and is not shown in other models. A second U6 structural
feature in our model which is not shown in other models
is a short stem-loop III. This stem-loop is only two
base-pairs long in many species, but is proven by obser-

J. C. Vaughn et al. / Open Journal of Animal Sciences 3 (2013) 36-48 43
vation of compensatory base mutations. We did however
fail to observe this stem-loop in the fungus C. albicans
and in E. histolyt i ca, showing that it is not universal.
3.2. The General Secondary Structure Model
is Not Universal and Multiple
Independent U4-snRNA Stem-Loop III
Losses Have Occurred During
Evolution
Representative structures for a diverse selection of
evolutionarily distant species show that the general
model is not universal. The most striking example is in
the absence of U4-snRNA stem-loop III (Figures 2(b),
(d), (e)), otherwise proven by observation of numerous
compensatory base changes. The absence of this stem-
loop has previously been noted in secondary structures
for various species of yeast and slime molds [12,25,32,
33]. It has been suggested that the absence of this
stem-loop is correlated with phylogenetic depth, imply-
ing that this structural feature was not present in the ear-
liest eukaryotes and is newly evolved [32]. We tested this
hypothesis by superimposing the presence/ absence of
this stem-loop onto the eukaryotic phylogenetic tree [31].
The results show that this stem-loop is present in all spe-
cies among the deeply-rooted flagellate Euglenozoa ex-
amined, but that three clearly independent secondary
losses have occurred during evolution (Figure 3). The
most recent is within Fungi, where all Ascomycete spe-
cies studied have lost the stem-loop, which is however
present in the Basidiomycete E. hasegawianum. An ear-
lier independent loss occurred among the Amoebozoa,
where the Mycetozoa slime mold species examined have
lost the stem-loop but the amoeboid Conosa E. histo-
lytica has not. The earliest loss is in the Alveolata, where
this feature is absent in the Apicomplexa P. falciparum
but not in the Cilliophora T. thermophila.
3.3. The General Secondary Structure Model
Compared to the Classical Y-Shaped
Model
It is informative to compare the secondary structures
of free U4- and U6-snRNAs with that of the duplex
which is formed upon their association during spli-
ceosome assembly, in consideration of the most parsi-
monious solution for their association (Figure 4). An
excellent free U4-snRNA secondary structure model has
previously been proposed based on the phylogenetic ap-
proach [32], utilizing a taxonomic diversity of species
extending only as deep as the slime mold Physarum. This
structure has been experimentally supported by the re-
sults of enzymatic digestion studies in rat U4-snRNA
[34]. The model contains four stem-loops, of which three
are incorporated directly into both our model and the
Y-shaped model. Stem-loop IV is disrupted in favor of
intermolecular base-pairing to form DS I, upon associa-
tion with U6-snRNA. Our resu lts confirm and extend the
previously proposed free U4-snRNA model, showing
that the structure has been retained to its origin within
the flagellate group Euglenozoa (Figure 3). There ar e no
differences in this part of our model in comparison to the
Y-shap ed model.
Previously proposed free U6-snRNA models for hu-
man [35] and yeast S. cerevisiae [36] show somewhat
differing structures, which are both supported by the re-
sults of chemical and enzymatic probing in these species
[37]. In the simplest free U6-snRNA secondary structure
model, as exemplified in Drosophila (Figure 1) and hu-
man, a short stem-loop is present at the 5’-end and the
entire 3’-terminus is folded into one long interrupted
stem-loop (Figure 4(a)). In human the chaperone p110,
an ortholog of Drosophila RNP-4F, has been shown to
bind primarily to free U6-snRNA nucleotides #38-57
[13], promoting unwinding of the long stem-loop and
base-pairing to two closely adjacent tracts on U4-snRNA,
which we have designated as DS I and DS II, followed
by chaperone release.
Our model and the Y- shaped model differ primarily in
how they show the U6-snRNA structure within the RNA
duplex. In the latter model, only the 5’-end stem-loop is
retained (Figure 4(c)), and no base-pairing occurs else-
where except within regions DS I and DS II, so that the
3’-end is unpaired. In our model, the base of old free
U6-snRNA is retained in stem-loop II, which brings the
3’-end into a duplex structure (Figure 4(b)). One set of
observations in support of this structure is seen in the
compensatory mutations present in this stem (Figure 1).
The results of previously reported chemical and enzy-
matic probing of the U4-/U6-snRNA duplex further
support the model which we have proposed and not the
Y-shaped model. In human, chemical reagent modifica-
tions were not observed within nucleotides #27-38 or
#94-106, which comprise the helix in stem-loop II in our
model but which are shown in long unpaired 5’- and
3’-tracts in the Y-shaped model. These observations are
indicative of a double-stranded structure here, and this
interpretation is confirmed by the observation of RNase
V1 cleavage 3’ to positions 33 and also 35 in the human
U4-/U6-snRNA duplex [37]. This is an enzyme which
cleaves specifically double-stranded RNA regions. These
results have also been reported by these authors upon
probing the base of free U6-snRNA stem-loop II. It has
been proposed that a potential third base-paired region of
contact may exist between U4- and U6-snRNA [24]. In
this view, the top of our U6-snRNA stem-loop II is
base-paired with a complement located within the long
single-stranded U4 connective between DS I and U4
stem-loop II. We are skeptical of this proposed third zone

J. C. Vaughn et al. / Open Journal of Animal Sciences 3 (2013) 36-48
44
Figure 3. Eukaryotic phylogenetic tree (31), showing taxonomic
distribution of species included in our study and stem-loops ob-
served. U4-snRNA stem-loop III has been independently lost at least
three times (arrows) during evolution of these RNAs.
Figure 4. Comparison between our general U4-/U6-snRNA secondary structure and the Y-shaped model. (a) Structures
of free U4- (32) and U6-snRNA (35) prior to their interaction. The primary position for binding of RRM in chaperone
RNP-4F/Prp24/p110 to free U6 stem-loop II (13) is indicated by heavy vertical overlay, and was determined experimen-
tally. The unwinding of U6 stem-loop II due to chaperone activity permits base-pairing between the two RNAs (region
bounded by the broken lines). The base of stem-loop II (cross-bars) remains associated in the resulting duplex structure
in our model. (b) Our general secondary structure model. (c) The Y-shaped model (12), shown inverted to facilitate
comparisons.

J. C. Vaughn et al. / Open Journal of Animal Sciences 3 (2013) 36-48 45
of RNA/RNA interaction, since different nucleotides in
the alignment must be utilized to create this structure.
For example, in S. cerevisiae and K. lactis the U4 region
of contact is very different from that for other species.
Within free U6-snRNA, nucleotides comprising stem-
loop III are contained within the long stem-loop (Figure
4(a)). The existence of stem-loop III in the duplex struc-
ture is proven by observation of compensatory base mu-
tations, but the stem length is reduced to only two base-
pairs in many species. Chemical and enzymatic probing
of the human U4-/U6-snRNA duplex [37] did not pro-
vide any further clarification for existence of this stem-
loop, since most of this region was contained in the site
of the primer utilized. Cryo-electron microscopy of iso-
lated U4-/U6-snRNA has been reported to show two
major structural domains linked by a thin connective [38],
in good agreement with our general secondary structure
model.
3.4. Phylogenetic Depth of the Genes
Encoding U4- and U6-snRNAs
The secondary structure of the U4-/U6-snRNA duplex
in our model is found to be identical, with the exception
of multiple independent losses of U4 stem-loop III dis-
cussed above, down to and including the deeply-rooted
flagellate group Euglenozoa. However, extensive BLAST
searches against both the Giardia [39] and Tricho monas
[40] genome sequences failed to detect any U4- or U6-
snRNA orthologs, using the corresponding T. brucei se-
quences as bait. The diplomonads and parabasalids are
generally considered to be descendants of the earliest
extant eukaryotes [31], leading us to consider the impli-
cations of this obser vat i on .
Success in BLAST searches is dependent on the de-
gree of nucleotide conservation between bait and prey
sequences, in addition to the completeness and accuracy
of the genomic sequence database itself. The nucleotide
sequences of genes encoding U6-snRNAs are among the
most highly conserved of any eukaryotic genes. For ex-
ample, the human and Drosophila U6-snRNA sequences
are 94% identical. The U6-snRNA sequence within and
immediately flanking the region of base-pairing with
U4-snRNA is exception ally well conserved. For example,
comparison between Drosophila and flagellate T. brucei
U6 nucleotides #40-75 shows 86% identity. This degree
of conservation is far greater than that observed for U4,
making identification of its most ancient orthologs more
difficult. It was therefore surprising that no U6-snRNA
genes turned up during BLAST searches against both the
diplomonad and p arabasalid g e nomes.
The Giardia and Trichomonas genome annotations are
well along, and we therefore asked if ANY of the U-se-
ries snRNA gene sequences have been annotated in these
species. Surprisingly, NONE of these genes have been
found despite an ~7X coverage during sequencing. In
addition, none of the genes encoding proteins which are
part of the U4- and U6-snRNPs in other eukaryotes have
been found (Steven Sullivan, personal communication).
Annotation of the Giardia genome has also failed to de-
tect any genes encoding U4- or U6-snRNA (Hilary Mor-
rison, personal communication). What are the implica-
tions of these observations? The spliceosome is widely
viewed as having evolved from self-splicing group II
introns like those in organellar protein-encoding genes as
well as in many bacteria [reviewed in 41,42], which do
not utilize the U-series of snRNAs. Interestingly, it has
been proposed that Giardia and Trichomonas nuclear
introns may represent evolutionary intermediates, show-
ing characteristics of both group II and spliceosomal
introns [43]. If so, then our study suggests that genes
encoding U4- and U6-snRNAs, and the resultant duplex
RNA which forms between them with a virtually identi-
cal secondary structure among all eukaryotes, may have
evolved within the flagellate group Euglenozoa.
3.5. A Potential Secondary RNP-4F
Chaperone Recognition Site in the
5’-UTR of Drosophila rnp-4f Pre-mRNA
May Play a Key Role in Controlling Its
Own Expression
We have previously described a long evolutionarily-
conserved potential stem-loop which arises by base-
pairing between all of the rnp-4f pre-mRNA intron 0 and
part of adjacent exon 2 in D. melanogaster [6,8]. We
have recently shown using RNA electrophoretic mobility
shift assay that retention of intron 0 within the rnp-4f
5’-UTR is correlated with binding of a dADAR protein
isoform, and that an unidentified second protein sus-
pected to be RNP-4F also binds to this stem-loop [9].
Subsequent work employing RNAi technology showed
that this dADAR protein is the truncated isoform [11].
We have proposed a negative feedback model for regu-
lating expression of rnp-4f mRNA under conditions of
RNP-4F excess within the developing fly central nervous
system [6]. If this hypothesis is correct, then the con-
served long stem-loop would be expected to contain a
nucleotide recognition sequence to which RNP-4F could
potentially bind, in competition with its preferred bind-
ing to a conserved sequence tract within the long stem-
loop of free U6-snRNA [13]. In Drosophila U6-snRNA
the conserved sequence contains nucleotides between
positions #38-5 7, although an even shorter sequen ce may
suffice for chaperone binding, but th is possibility has not
yet been tested. Examination of the Drosophila con-
served rnp-4f 177-nt stem-loop nucleotide sequence/
structure shows that a 12-nt tract closely resembling the
preferred U6-snRNA binding site is indeed present (Fig-
re 5(b), (c)). An additional similarity between the u

J. C. Vaughn et al. / Open Journal of Animal Sciences 3 (2013) 36-48
46
(a)
(b)
(c)
Figure 5. A 177-nt long Drosophila rnp-4f stem-loop in the pre-mRNA 5’-UTR regulatory region contains a potential RNP-4F pro-
tein chaperone binding site. (a) Orientation diagram showing position of long stem-loop which forms by hydrogen bonding between
intron 0 and part of exon 2. (b) Long interrupted rnp-4f stem-loop secondary structure as predicted from Mfold program (29). The 5’-
and 3’-limits of intron 0 are indicated, in addition to alternative 3’-splice site within exon 2 (8) and evolutionarily-conserved short
stem-loop (boxed) at tip of the longer structure (6). The highlighted nucleotides near the tip show position of potential RNP-4F pro-
tein binding site postulated to compete with the preferred experimentally determined tract within U6-snRNA (13). (c) Alignment at
region of chaperone RNP-4F/Prp24/p110 binding site to U6-snRNA in various species, and to potential rnp-4f pre-mRNA nucleo-
tides.
RNP-4F chaperone substrate free U6-snRNA (Figure
4(a)) and rnp-4f pre-mRNA is that in both cases the rec-
ognition sequence is con tained within a long, interrupted
stem-loop structure. In Drosophila free U6- snRNA this
stem-loop contains 81-nt, while in rnp-4f the stem-loop
contains 177-nt. Finally, RNP-4F is a nuclear protein (6)
and thus would be expected to have access to the long
stem-loop in rnp-4f pre-mRNA. These observations sup-
port the hypothesis that excess RNP-4F protein may
competitively bind to a 5’-UTR regulatory region within
its own pre-mRNA, playing a role in negative feedback
control.
4. CONCLUSION
Our long standing interest in Drosophila splicing as-
sembly factor RNP-4F, which functions as a chaperone
to facilitate bonding between U4- and U6-snRNA, led us
to analyze the secondary structure of the U4-/U6-snRNA
duplex. Close study of published chemical and enzy-
matic probing results on th e proposed human and yeast S.
cerevisiae U4-/U6-snRNA structures [37] suggested to us
certain inconsistencies within the classical Y-shaped mo-
del [12]. Further, preliminary comparison of the clas-
sical model with a computer-generated secondary struc-
ture also revealed inconsistencies, which led us to re-
examine this model. We deemed this timely in light of
the many new U4-and U6-snRNA sequences that have
become available, in large part, by recent genomic se-
quencing projects. Our study, utilizing the comparative
phylogenetic approach, eventually resulted in a revised
and improved U4-/U6-snRNA secondary structure model.
The model proven by observation of abundant com-
pensatory base mutations in every stem is shown to be
general but not universal, and structural variations have
been traced to their origins within the phylogenetic tree.
We have extensively probed the eukaryotic tree to its
deepest roots, and our results suggest that U4- and U6-

J. C. Vaughn et al. / Open Journal of Animal Sciences 3 (2013) 36-48 47
snRNAs apparently evolved after the emergence of lines
leading to the diplomonad Giardia and the parabasalid
Trichomonas, but once established they have maintained
a remarkably well conserved U4-/U6-snRNA secondary
structure extending to, and including, the flagellates
among the Euglenozoa. An unexpected result of this
study was discovery of a potential competitive binding
site for Drosophila splicing assembly factor RNP-4F to a
5’-UTR regulatory region within its own pre-mRNA,
which may play a role in negative feedback control [6].
This negative feedback expression control model awaits
experimental testing.
5. ACKNOWLEDGEMENTS
We wish to thank the late Carl Woese for his helpful discussions of
RNA secondary structure determinations over more than two decades,
to whom this study is dedicated. We also thank Jane Carlton and Steven
Sullivan for helpful discussion bearing on Giardia and Trichomonas
splicing machinery components. Peter Dobler is to be thanked for his
help in constructing the secondary structure figures in the manuscript.
This work was primarily supported by National Institutes of Health
(NIH) Grant 1-R15-GM070802-01 and 1-R15-GM093895-01 to J.
Vaughn.
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