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Contents lists available at ScienceDirect
Seminars in Cell & Developmental Biology
j ournal homepage: www.elsevier.com/locate/semcdb
Review
Minor spliceosome and disease
1 ∗
Bhupendra Verma , Maureen V. Akinyi, Antto J. Norppa, Mikko J. Frilander
Institute of Biotechnology, PL56 (Viikinkaari 5), 000014, University of Helsinki, Finland
a r t i c l e i n f o a b s t r a c t
Article history: The U12-dependent (minor) spliceosome excises a rare group of introns that are characterized by a
Received 4 July 2017
highly conserved 5 splice site and branch point sequence. Several new congenital or somatic diseases
Received in revised form
have recently been associated with mutations in components of the minor spliceosome. A common
21 September 2017
theme in these diseases is the detection of elevated levels of transcripts containing U12-type introns,
Accepted 27 September 2017
of which a subset is associated with other splicing defects. Here we review the present understanding
Available online xxx
of minor spliceosome diseases, particularly those associated with the specific components of the minor
spliceosome. We also present a model for interpreting the molecular-level consequences of the different Keywords: diseases.
U12-type introns
© 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license
Minor spliceosome (http://creativecommons.org/licenses/by/4.0/). Human diseases
Pre-mRNA splicing
Cryptic splice sites
Exon skipping
Intron retention
Contents
1. Introduction ...... 00
2. Minor spliceosome in human disease ...... 00
2.1. Diseases affecting the intron recognition step ...... 00
2.1.1. Isolated growth hormone deficiency ...... 00
2.1.2. Cerebellar ataxia ...... 00
2.1.3. Myelodysplastic syndrome ...... 00
2.2. Diseases affecting the formation of catalytically active spliceosome ...... 00
2.2.1. MOPD1/TALS...... 00
2.2.2. Roifman syndrome ...... 00
2.3. Diseases with mutations in the U12-type splice sites ...... 00
2.4. Other spliceosome mutations affecting minor spliceosome activity ...... 00
3. Minor spliceosome components as biomarkers ...... 00
4. Consequences of minor spliceosome-specific mutations ...... 00
4.1. The fate of the mRNA: intron retention vs cryptic splicing ...... 00
4.2. Fate of the mRNA and disease severity ...... 00
4.3. Defects with individual genes vs a global splicing defect ...... 00
5. Future perspective ...... 00
Acknowledgements...... 00
References ...... 00
∗
Corresponding author.
E-mail address: [email protected] (M.J. Frilander).
1
Present address: Department of Biotechnology, All India Institute of Medical Sciences, New Delhi, India.
https://doi.org/10.1016/j.semcdb.2017.09.036
1084-9521/© 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Please cite this article in press as: B. Verma, et al., Minor spliceosome and disease, Semin Cell Dev Biol (2017), https://doi.org/10.1016/j.semcdb.2017.09.036
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1. Introduction U11/U12 di-snRNP protein component, Urp/ZRSR2 takes up the role
of 3 ss recognition with minor introns [19]. Following this initial
Pre-mRNA splicing is an essential step in the gene expression recognition, both splicing pathways proceed with association of a
pathway of all eukaryotes. During splicing, the non-coding intron tri-snRNP, either U4atac/U6atac.U5 or U4/U6.U5 (Fig. 1B; [20,21]).
sequences are recognized and removed from precursor mRNA (pre- Further rearrangements in RNA–RNA and RNA–protein interac-
mRNA) by the spliceosome, a large ribonucleoprotein complex. tions lead to the formation of a catalytically active spliceosome and
Defects in splicing of one or more mRNA species are a major cause catalytic excision of the intron [20,22,23].
of human diseases and present estimates suggest that up to 60%, Here, we review the role of the minor spliceosome in human
are associated with pre-mRNA splicing [1,2]. In addition to mono- diseases, with a specific focus on diseases caused by mutations
genic disorders, genetic variants altering splicing are also thought in the integral components of the minor spliceosome. Given that
to be an important contributor to complex diseases and cancer most protein components and U5 snRNA are shared with the major
[3,4]. The majority of disease-associated splicing defects are cis- spliceosome, there are also several diseases where mutations dis-
acting mutations within a single gene that disrupt either the splice rupting shared components can potentially affect the functions of
site consensus sequences or splicing regulatory elements located in both spliceosomes. Those have been discussed in detail elsewhere
introns or exons [5]. Notably, exonic mutations interpreted as mis- [24].
sense, nonsense or silent also commonly affect splicing [4,6,7]. The
outcome of cis-acting splicing mutations is often either a formation
of abnormal or non-functional protein species or accelerated decay 2. Minor spliceosome in human disease
of the affected individual mRNAs. In contrast, mutations in splicing
factors, spliceosome components or spliceosome assembly factors The direct targets for human diseases specific for the minor
often lead to widespread defects in the processing of large numbers spliceosome are the unique snRNA and protein components. This
of pre-mRNAs [8–10]. includes several components of the U11/U12 intron recognition
Most metazoan species contain two distinct pre-mRNA splicing complex and the U4atac and U6atac snRNAs in the minor tri-snRNP.
machineries known as the major (U2-dependent) and minor (U12- The U11/U12 di-snRNP contains, in addition to the U11 and U12
dependent) spliceosomes, which recognize and excise either the snRNAs, seven integral proteins (65K, 48K, 59K, 35K, 31K, 25K and
major (U2-type) or minor (U12-type) class of introns, respectively. 20K) that are unique to the minor spliceosome [25,26]. Addition-
In contrast to the major introns, U12-type introns are character- ally, the Urp/ZRSR2 protein associated with the U11/U12 di-snRNP
ized by divergent and highly conserved 5 splice site (5 ss) and has been reported to function in both minor and major spliceo-
branch point sequences (BPS) (Fig. 1A; [11]). These introns also somes, with an essential role for U12-type intron 3 ss recognition
lack the characteristic polypyrimidine tract (PPT) that is present in [19].
U2-type introns immediately upstream of the 3 splice site (3 ss). To date five human diseases with mutations in the spe-
∼
Minor introns constitute only 0.35% of all human introns and have cific components of the minor spliceosome have been described
been reported to be present in 700–800 genes, each of which typ- (Table 1). Three of them affect the components of the U11/U12 di-
ically carry only a single U12-type intron and multiple U2-type snRNP, namely the U11/U12-65K protein (RNPC3; [27]), U12 snRNA
introns. U12-type introns are enriched in genes that represent a (RNU12; [28]) and Urp protein (ZRSR2; [29]); while two diseases are
rather restricted set of functional classes and pathways. Particu- attributed to mutations in the U4atac snRNA (RNU4ATAC; [30–32]).
larly they are present in genes related to ‘information processing Each of these diseases is hypomorphic, leading only to a partial loss
functions’, such as DNA replication and repair, transcription, RNA of minor spliceosome function because correctly spliced mRNAs
processing, and translation, but can also be found in genes related can be detected in the patient cells. We briefly introduce each
to cytoskeletal organization, vesicular transport, and voltage-gated disease and later discuss the impact of disease mutations on the
ion channel activity, as suggested originally by Burge et al., [12] assembly of the minor spliceosome and the fate of affected mRNAs.
and verified later [13,14]. Both the identities of genes carrying U12- Apart from mutations in the core minor spliceosome compo-
type introns and their positions within the genes are evolutionarily nents, we also describe the few reported human diseases caused
conserved [15]. by mutations at the splice sites of U12-type introns, though we
The overall organisational and functional features of both note that this appears to be underexplored territory given the small
spliceosomes are highly similar. Both are composed of five small number of cases reported so far. Finally, we mention the emerging
nuclear RNA (snRNA) molecules that associate with protein factors role for the minor spliceosome in cancer and autoimmune disorders
to give rise to small nuclear ribonucleoproteins (snRNPs). Within as well as neurodegenerative diseases.
the minor spliceosome, four of the five snRNAs are unique. Specifi-
cally, U11, U12, U4atac, and U6atac replace the major spliceosome
counterparts U1, U2, U4 and U6 snRNAs, respectively. U5 snRNA 2.1. Diseases affecting the intron recognition step
is shared between the two spliceosomes. Of the 200–300 pro-
teins associated with spliceosomes, most are thought to be shared 2.1.1. Isolated growth hormone deficiency
between the two systems and only 7 proteins, associated with U11 Recessive mutations in the RNPC3 gene, encoding U11/U12-
and U12 snRNPs, are unique to the minor spliceosome [16,17]. 65K, one of the seven minor spliceosome-specific proteins, have
The highly similar spliceosome composition is reflected in been associated with isolated growth hormone deficiency (IGHD)
the conserved assembly pathway and catalytic mechanism. Both and associated pituitary hypoplasia. The 65K protein is part of a
spliceosomes are assembled sequentially starting from intron molecular bridge that connects the U11 and U12 snRNPs into a di-
recognition, followed by formation of a catalytically active spliceo- snRNP (Fig 1B; [33]). Initially, RNPC3 mutations were detected in a
some and joining of the exons flanking the excised intron. With single family only [27], but additional cases with overlapping muta-
minor introns, the 5 ss and BPS are co-operatively recognized by tions and similar phenotypes have been described subsequently
a pre-formed U11/U12 di-snRNP [18], contrary to the sequential [34]. IGHD is a condition characterized by a shortage or absence
recognition of these sequences by individual U1 and U2 snRNPs of growth hormone, with the absence of associated pituitary hor-
in major introns. Since the PPT is lacking in minor introns, the mone deficiencies. Genetically, IGHD is a diverse disease and can
U2AF1/2 heterodimer that recognizes the PPT and 3 ss of major result from either recessive or dominant mutations in various genes
introns does not associate with minor introns. Instead, an integral involved in pituitary development or function [35].
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Fig. 1. (A) Consensus sequences of human U12-type and U-2 type introns. Adapted from [11]. (B) A simplified assembly pathway of the U12-dependent spliceosome
highlighting the intron recognition and catalytic stages of the assembly. The schematic structure of the U11/U12 intron recognition complex includes protein components
specific to the U12-dependent spliceosome (65K (RNPC3), 59K (PDCD7), 48K (SNRP48), 35K (SNRNP35), 31K (SNRNP31/ZCRB1), 25K (SNRP25K), 20K (SNRNP20/ZMAT5), Urp
(ZRSR2) and also the SF3b complex shared between the two spliceosomes. Additionally, human diseases specifically associated with either assembly step of the minor
spliceosome assembly are indicated on right.
All reported IGHD cases associated with RNPC3 mutations are terminal RNA recognition motif (RRM) of the protein, while the
compound heterozygous with a missense P474T mutation com- R205X maps to the proline-rich region between the two RRMs
bined with either R502X or R205X nonsense mutation [27,34]. At (Fig. 2A). Although the tissue affected in IGHD, the pituitary gland,
the protein level the P474T and R502X mutations map to the C- cannot be sampled from the patients, RT-PCR and RNA-seq analyses
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Table 1
Human diseases showing defects in splicing of U12-type introns.
Defect in Locus Disease Abbreviation References
Integral components of RNPC3 Isolated Growth Hormone Deficiency IGHD [27]
minor snRNPs ZRSR2 Myelodysplastic Syndrome MDS [29]
RNU12 Early-onset Cerebellar ataxia EOCA [28]
RNU4ATAC Microcephalic osteodysplastic primordial dwarfism/Taybi-Linder Syndrome MOPD1/TALS [31,32]
RNU4ATAC Roifman syndrome RFMN [30]
U12-type 5 ss STK11 Peutz-Jegher’s syndrome PJS [49]
TRAPPC2 Spondyloepiphyseal dysplasia tarda SEDT [50]
Biogenesis/Assembly FUS Amyotrophic lateral sclerosis ALS [64]
SMN1 Spinal muscular atrophy SMA [10,57–61]
Fig. 2. Disease-associated mutations in the specific protein and snRNA components of the minor spliceosome.
A) Domain structure of the U11/U12-65K proteins with the locations of IGHD-associated mutations indicated.
B) Secondary structure of U12 snRNA showing the location of a point mutation associated with EOCA. Binding site for the U11/U12-65K protein is also indicated.
C) Secondary structure of the U4atac/U6atac di-snRNA complex showing mutations associated with MOPDI (red) and RFMN (cyan). A mutation shared between MOPD1/TALS
and RFNM patients is indicated in red text surrounded by a cyan rectangle. Binding sites for 15.5K, PRPF31, the PRPF4/PRPF3/PPIH complex proteins and the U11/U12-65K
protein are also shown.
D) Domain structure of the ZRSR2 protein showing mutations associated with MDS. Mutations are from the COSMIC database [76]. Mutations studied by Madan et al. [29]
are shown in blue.
was carried out on P474T/R502X patient lymphocytes [27]. Consis- as well as reduced stability of the U11/U12 di-snRNP complex.
tent with the function of the 65K protein in the intron recognition This result is consistent with the function of the 65K protein as a
complex, several types of aberrant splicing events associated with component of themolecular bridge between U11 and U12 snRNPs
a subset of U12-type introns were detected, including increased through interactions with the U11-59K protein and U12 snRNA
retention of U12-type introns, activation of nearby cryptic U2- [33]. The 65K-U12 snRNA interaction is mediated by the C-terminal
type splice sites and exon skipping events. Biochemical analysis RRM domain and, as predicted, the IGHD mutations were shown
of patient cells revealed a reduction in levels of the 65K protein, to either reduce (P474T) or eliminate (R502X) the binding of 65K
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to the stem-loop III of U12 snRNA (Fig. 2B; [36]). The picture that domain (UHM) flanked on each side by a CCCH-type zinc finger, and
is emerging from these studies is that the IGHD mutations lead an RS domain near the C-terminus (Fig. 2D). ZRSR2 is an integral
to impaired interaction between the 65K protein and U12 snRNA, component of the U11/U12 di-snRNP and functions in the recogni-
which in turn causes destabilization or reduced formation of the tion of the U12-type 3 splice site, but it has also been reported to
U11/U12 di-snRNP. Additionally, it is likely that both RNPC3 non- function during the second catalytic step of U2-type intron splicing
sense mutations described for IGHD lead to allele-specific decay of [19,25]. MDS-associated mutations in ZRSR2 include a wide spec-
the 65K mRNA via nonsense mediated decay (NMD) pathway, as trum of nonsense, missense, frameshift and splice site mutations
shown recently for the R502X allele [36]. This is expected to signif- which are uniformly scattered across the gene (Fig. 2D). This distri-
icantly reduce the levels of 65K proteins encoded by the nonsense bution is in striking contrast to MDS cases with mutations in U2AF1,
alleles. SRSF2 and SF3B1 genes that show a narrower mutation spectrum,
However, we note that additional mechanisms may be at play, with particular preference for missense mutations that are located
as it is not currently known whether the 65K protein is purely a mostly within a small number of hotspot positions.
structural component of the U11/U12 di-snRNP, or whether it has Of the numerous somatic mutations reported in the ZRSR2
additional functions in the splicing process. A zebrafish study indi- (Fig. 2D), only few have been studied in detail [29]. In the Madan
cated that the 65K protein may have other functions later in the et al. [29] RNAseq study, bone marrow samples from eight ZRSR2-
spliceosome assembly [37]. Consistently, recent work reported an mutant MDS patients, each carrying a different mutation, were
interaction between 65K and the 3 terminal stem-loop of U6atac compared to both ZRSR2 wild-type MDS patients and healthy indi-
snRNA (Fig. 2C; [38]), which is similarly impaired by the IGHD viduals. The analysis revealed a global increase in U12-type intron
mutations [36]. retention, as well as activation of cryptic U2-type splice sites only in
MDS patients carrying the mutated ZRSR2 allele. Importantly, the
2.1.2. Cerebellar ataxia effect of ZRSR2 mutations and knockdown of ZRSR2 on U2-type
A mutation in the noncoding U12 snRNA gene (RNU12) has intron splicing were comparatively small. The affected U2-type
recently been associated with an early-onset cerebellar ataxia introns were mostly in genes containing U12-type introns. Similar
(EOCA) [28]. In general, ataxias are a diverse group of disorders conclusions have also been reported in plants [41]. These findings
characterized by defects in muscle coordination which, in the case are in stark contrast to the earlier biochemical experiments which
of cerebellar ataxias, results from abnormal development and/or suggested that an essential role for ZRSR2 was in splicing of both
degeneration of the cerebellum. The patients in this single study U2- and U12-type introns [19].
are homozygous for a point mutation (84C > T; see Fig. 2B) with
symptoms of hypotonia at infancy, delayed motor development, 2.2. Diseases affecting the formation of catalytically active
abnormal gait, speech and learning difficulties [28]. RNAseq and spliceosome
RT-PCR analyses of patient mononuclear cells showed increased
U12-type intron retention and upregulation of the U12 snRNA. 2.2.1. MOPD1/TALS
The 84C > T mutation is located at the base of U12 snRNA stem- Recessive mutations in the U4atac snRNA gene (RNU4atac)
loop III, and is predicted to weaken the stem-closing G-C base-pair associated with MOPD1/TALS, a rare autosomal recessive disease,
by converting it to a weaker G-U base-pair (Fig. 2B). Mechanistic represent the first human disease with mutations in a specific
understanding of how this mutation leads to a defect in minor component of the minor spliceosome [31,32,42]. U4atac snRNA
spliceosome function is currently lacking. The mutation does not is an essential component of the U4atac/U6atac.U5 tri-snRNP
directly overlap with any known functional elements, such as (minor tri-snRNP), which joins the nascent spliceosome after the
sequences involved in known RNA–RNA or RNA–protein interac- intron boundaries have been recognized by the U11/U12 di-snRNP
tions; however, the apical part of this long stem-loop acts as the (Fig. 1B). During the assembly of the minor tri-snRNP, U4atac forms
binding site for the 65K protein (Fig 2B; [33]) and the assembly site an extensively base-paired duplex with the U6atac snRNA (Figs. 1B
for the Sm protein ring, an integral part of all snRNPs, is located and 2C). This U4atac/U6atac di-snRNA complex forms a platform
only two nucleotides away. Furthermore, as the U12 snRNA not for the binding of tri-snRNP-specific proteins that are needed for
only recognizes the branch point sequence during intron recogni- the association of the U5 snRNP to the complex.
tion, but is also part of the catalytic core of the minor spliceosome, MOPD1/TALS in its severe form is characterized by intrauterine
several additional mechanistic scenarios are conceivable. Further and postnatal growth retardation, developmental defects in sev-
functional studies are needed to uncover the mechanistic basis of eral organs, including microcephaly, and death typically within 3
this disease. years after birth. However, milder forms of the disease have been
reported displaying more subtle growth retardation, less severe
2.1.3. Myelodysplastic syndrome developmental defects and/or survival to at least to 12 years of age
Myelodysplastic syndrome (MDS; myelodysplasia) is a term for or even adulthood [43,44]. Presently, individual single-nucleotide
a group of disorders characterized by inefficient hematopoiesis, point mutations at ten different positions within the U4atac snRNA
with a risk of progression to acute myeloid leukemia (AML). have been associated with MOPD1/TALS (Fig. 2C); in addition, one
In recent years, several whole-exome sequencing studies have patient with a duplication of U4atac nucleotides 16–100 has been
revealed frequent somatic mutations in genes encoding splicing reported [44]. All patients are either homozygous or compound
factors, including U2AF1, ZRSR2, SRSF2 and SF3B1 in patients with heterozygous for the disease mutations. In contrast, heterozy-
MDS reviewed by [9,39]. Although patients with a combination of gous parents carrying only a single disease-causing mutation are
different splicing factor mutations have been reported, typically phenotypically normal, suggesting that a loss of a single allele is
only one splicing factor gene in each patient has been mutated. Fur- well-tolerated at the cellular level.
thermore, the splicing factor mutations frequently co-occur with The majority of disease-causing mutations, particularly those
mutations in genes encoding epigenetic factors, cell signaling reg- leading to severe forms of the disease, are found in the U4atac
ulators and transcriptional regulators [39,40]. Here, we focus on the 5 stemloop, with only few pathogenic mutations located else-
Urp/ZRSR2 protein involved in U12-type 3 ss recognition. where in the U4atac snRNA (Fig. 2C). Earlier studies from the major
The ZRSR2 protein shares both primary structure and functional spliceosome have indicated that this region is recognized by a group
features with the splicing factor U2AF1, that facilitates 3 ss recogni- of proteins (Fig. 2C), particularly the 15.5K (Snu13) and PRPF31 pro-
tion with the major introns. Both contain a central U2AF-homology teins, that are both necessary for the association of U5 snRNP to
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the tri-snRNP [45]. Consistently, the MOPD1/TALS mutations in this These three cases do not only demonstrate that a single point
region reduce the 15.5K binding, which in turn decreases the cellu- mutation in the highly conserved U12-type 5 ss sequence can
lar levels of U4atac/U6atac.U5 tri-snRNP [46]. Importantly, there is efficiently disable the U12-type intron recognition, but also that
a correlation between disease severity and the affinity of the 15.5K splicing of a U12-type intron is linked to other splicing events in
protein. Mutations on the tip of the 5 stemloop that reduce most the same transcript and that in some cases the outcome may be
the 15.5K binding, also lead to severe forms of the disease. In con- difficult to predict. PJS, in which a mutation at the +1 position
trast, the more distal mutations (such as U4atac positions 30, 53 and of an AT–AC-type U12-type intron is changed to G, is a particu-
55–see Fig. 2C) have little effect on the binding of the 15.5K pro- larly illuminating case [49]. At the sequence level, it appears to
tein and are consequently associated with milder symptoms, such be a simple U12-type 5 ss subtype change, but the experimentally
as longer life expectancy [43,46]. Additionally, at least one of the determined outcome is in fact complex, showing activation of sev-
mutations (124G > A) reduces the U4atac snRNA expression levels eral non-canonical 3 splice sites that illuminate the flexibility of
[46]. the U12-dependent spliceosome in 3 ss recognition. Even though
No transcriptome studies have yet been conducted for the outcome in each case is the same, i.e. introduction of a pre-
MOPD1/TALS, but qPCR analyses have shown increased U12-type mature STOP-codon and mRNA decay via the NMD pathway, this
intron retention in patient cells that can be rescued by expression particular mutation demonstrates that simple point mutations can
of wild type U4atac snRNA [31,32]. Significantly, an analysis with lead to unexpected consequences.
a reporter system indicates that >90% of the minor spliceosome
activity may have been compromised [31]. However, it is possible
that the reporter system used may have exacerbated the splic-
ing defect as the endogenous transcripts in patient cell lines and
2.4. Other spliceosome mutations affecting minor spliceosome
iPS cells carrying the mutation show much milder splicing defects
[31,32,46]. activity
In addition to diseases caused by mutations in the specific
2.2.2. Roifman syndrome
components of the minor spliceosome, there are several addi-
In addition to MOPD1/TALS, mutations in the RNU4atac locus
tional human diseases caused by congenital or somatic mutations
have been associated with a phenotypically different Roifman
in either the shared components of the two spliceosomes or the
syndrome (RFMN) [30]. Patients suffering from RFMN are char-
assembly factors necessary for both major and minor snRNPs.
acterized by poor pre- and post-natal growth, distinct facial
Examples of the former include Retinitis pigmentosa (RP) mutations
dystrophies, cognitive delay and immunological defects which,
in genes encoding the tri-snRNP specific proteins PRPF3, PRPF4,
with the exception of growth defects, are distinct to Roifman
PRPF6, PRPF8, PRPF31 and BRR2 [Reviewed in 8], and MDS muta-
patients [30]. Interestingly, all RFMN patients are compound het-
tions in PRPF8 [52], in SF3b1, a component of both U2 snRNP and
erozygotes, with one mutation shared with MOPD1/TALS patients
U11/U12 di-snRNP [26], and in SRSF2, a splicing activator poten-
with severe forms of the diseases (Fig. 2C). The other mutation is
tially affecting both spliceosomes [53]. Spinal muscular atrophy
predominantly located among the highly conserved positions of the
(SMA) represents the latter group and is caused by mutations in
RNU4atac stem II, except for a single case with a mutation within
the SMN1 gene, coding for an essential factor for the assembly of all
the Sm site.
Sm-class snRNPs, including components of both major and minor
No functional studies have been performed with the RFMN
spliceosomes [54].
mutations, but it is safe to assume that the mutations shared with
With RP, the shared protein composition between the minor
the MOPD1/TALS lead to a similar defect in minor tri-snRNP assem-
and major tri-snRNPs [17] and functional studies [55,56] predict
bly [46]. In contrast, the stem II mutations presumably lead to a
that the mutations should have an equal effect on both the major
milder functional defect in minor splicing, given the overall less
and minor tri-snRNPs. With SMA, however, several investigations
severe disease phenotype. While the molecular defects resulting
using different model systems have reported a preferential reduc-
from the stem II mutations remain to be determined, the investiga-
tion in the cellular levels of minor snRNPs [10,57–61]. In a subset of
tion from the major spliceosome assembly tentatively suggest that
the studies this has been reported to lead to an increased retention
the association of the PRPF4/PRPF3/PPIH complex (Fig. 2C) with the
U12-type introns, suggesting that defects in the splicing of minor
stem II may be compromised [30,47,48].
introns may contribute to the SMA symptoms. In a Drosophila model
In addition, RNAseq analyses of the patient cells revealed a
of SMA the effects on neuronal cells were attributed to a single
widespread retention of U12-type introns. Significantly, neither
U12-type intron containing gene, Stasimon, encoding a transmem-
increased retention of U2-type introns, nor other types of alter-
brane protein required for axonal growth and regulation of synaptic
native splicing changes were detected in the patient cells [30],
transmission of motor neurons [60]. However, some aspects of this
suggesting a very specific splicing defect affecting U12-type introns
only. study have been challenged subsequently [62].
Finally, two independent studies have linked minor spliceo-
some to amyotrophic lateral sclerosis (ALS). Ishihara et al. [63]
2.3. Diseases with mutations in the U12-type splice sites
reported that ALS-associated depletion of TDP-43 protein leads to
a co-depletion of U12 snRNA both in cultured cells and in patient
To our knowledge there are only two reported cases in lit-
samples, presumably due to defects in snRNP assembly. In another
erature that specifically attribute U12-type splice site mutations
study [64], ALS-associated mutations in the Fused in sarcoma (FUS)
with human disease. These include the autosomal dominant dis-
protein lead to reduced nuclear import of FUS and its accumulation
order Peutz-Jeghers syndrome (PJS), characterized by the presence
in the cytoplasm. FUS is a multifunctional RNA-binding protein that
of multiple intestinal polyps and a high risk for cancer [49], and
interacts with several splicing factors, including U11 snRNP and is
Spondyloepiphyseal dysplasia tarda (SEDT), an X-linked recessive
needed for the splicing of at least a subset of minor introns [64]. Sig-
disorder [50]. Both are caused by mutations in a U12-type 5 ss that
nificantly, the cytoplasmic accumulation of a particular FUS mutant
lead to incorrect splicing of the respective mRNAs. Additionally, the
leads to a concomitant mislocalization of U11 and U12 snRNPs in
Motor endplate disease in mouse reports a mutation of a U2-type
the cytoplasm, which in turn leads to defects in the splicing of
5 ss in a downstream intron, leading to skipping of the upstream
U12-type introns.
U12-type intron [51].
Please cite this article in press as: B. Verma, et al., Minor spliceosome and disease, Semin Cell Dev Biol (2017), https://doi.org/10.1016/j.semcdb.2017.09.036
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Fig. 3. Model for molecular consequences of the minor spliceosome diseases. Mutations in the minor spliceosome components can lead to nuclear retention of the partially
spliced mRNA, or intron retention and associated activation of cryptic U2-type splice sites or exon skipping events. These in turn lead to nuclear or cytoplasmic decay, or
formation of the abnormal proteins as indicated.
3. Minor spliceosome components as biomarkers molecular consequences of the mutations, but may also help to
interpret the subsequent disease phenotypes.
In addition to being a target for disease-causing mutations, sev-
eral components of the minor spliceosome have been reported as
4.1. The fate of the mRNA: intron retention vs cryptic splicing
cancer biomarkers. A recent study by Xu et al. [65] using serum
from patients with scleroderma and coincident cancer reported
According to this classification the mutations associated with
autoantibodies targeted to components of the U11/U12 di-snRNP,
both the MOPD1/TALS and RFMN affect the formation of the
particularly the U11/U12-65K protein, but also the 25K, 35K and
U4atac/U6atac.U5 tri-SNRP (see 2.2.1 and 2.2.2), but not the initial
59K proteins. Of these, the U11/U12-65K autoantibodies appear
intron recognition step. Therefore, in these diseases the spliceo-
to show a reliable association with an increased risk of cancer
some assembly is arrested at the pre-spliceosome stage where
occurring within 2 years of the onset of scleroderma [66]. Similar
the U11/U12 di-snRNP is bound to the intron but the subsequent
associations have been described for U11 snRNA in familial prostate
assembly steps are inhibited. Consequently, the bound U11/U12
cancers [67] and U11-59K protein in Acute Myeloid Leukemia [68].
di-snRNP can be expected to maintain exon-definition interactions
However, the role of minor spliceosome-specific autoantibodies in
with the surrounding (U2-type) introns via the RS domain contain-
the pathogenesis of scleroderma and/or coincident cancer, or the
ing U11-35K [69] and possibly Urp/ZRSR2 proteins. Furthermore,
expression level changes of other minor spliceosome components
the U11/U12 di-snRNP bound to the partially spliced mRNA can trap
in other diseases is presently unclear.
such transcripts in the nucleus [70], where they may be targeted
by the nuclear quality control mechanisms [71,72].
In contrast, the loss of U11/U12 binding and the recognition of
4. Consequences of minor spliceosome-specific mutations U12-type introns in IGHD and MDS (and possibly EOCA) leads to a
different outcome. Due to the loss of U11/U12 binding, transcripts
Presently, mutations in four specific components of the minor containing unspliced U12-type introns are free to be exported to the
spliceosome have been associated with human diseases showing cytoplasm. Concomitantly, the loss of local exon definition interac-
very different disease phenotypes. RT-PCR and RNAseq analyses tions may lead to increased activation of cryptic splice sites or exon
have demonstrated that each of the five human diseases show skipping events near the U12-type introns [73]. The outcome can
the expected molecular level phenotype, i.e. the increased levels be either truncated or altered protein, or mRNA decay via the NMD
of unspliced U12-type introns in the patient cells. With a subset of quality control pathway (Fig. 3).
diseases, additional splicing defects, particularly activation of cryp- Is there evidence supporting this model? RNAseq analyses from
tic U2-type splice sites and exon skipping events have also been both IGHD and MDS patient cells provide extensive evidence of acti-
described. Presently, there has not been clear consensus on how to vation of cryptic U2-type splice sites and increased levels of exon
interpret the molecular consequences of minor spliceosome muta- skipping near U12-type introns in addition to the expected reten-
tions. Here, we propose a simple classification of disease-causing tion of U12-type introns [27,29]. In contrast, besides the increased
mutations according to their impact on the recognition of the U12- intron retention of U12-type introns seen with both MOPD1/TALS
type introns. This provides a simple framework for predicting the and RFMN, there is no evidence of the associated cryptic U2-type
Please cite this article in press as: B. Verma, et al., Minor spliceosome and disease, Semin Cell Dev Biol (2017), https://doi.org/10.1016/j.semcdb.2017.09.036
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8 B. Verma et al. / Seminars in Cell & Developmental Biology xxx (2017) xxx–xxx
splicing events [30–32,46] as stated by Merico et al. [30] in their significance and function of the U12-dependent spliceosome. How-
detailed RNAseq analysis of RFMN patient cells. Together, present ever, many aspects of the disease process, including the basis of the
evidence supports the proposal that the consequences of the minor tissue specificity and the detailed mechanism of the disease process
spliceosome mutations are linked to the type of assembly defect are yet to be determined for most diseases, requiring animal models
introduced. However, with both MOPD1/TALS and RFMN it is pos- of each disease. These will also help to address the more fundamen-
sible that some arrested transcripts trapped in the nucleus may tal question of the existence of two separate spliceosomes.
either lose the bound U11/U12 di-snRNP or undergo the splicing
process post-transcriptionally (see Fig. 3). Acknowledgements
4.2. Fate of the mRNA and disease severity
This work was supported by the Academy of Finland (Grant
140087 to MJF and Grant 278798 to BV) and Sigrid Jusélius Foun-
It is somewhat puzzling to note that the severity of congen-
dation (MJF). AJN was supported by the Integrative Life Science
ital minor spliceosome diseases and the level of mRNA splicing
doctoral program at the University of Helsinki.
defect follow a counterintuitive association as described above.
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