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Non-coding RNAs: lessons from
the small nuclear and small
nucleolar RNAs
A. Gregory Matera*, Rebecca M. Terns‡ and Michael P. Terns‡
Abstract | Recent advances have fuelled rapid growth in our appreciation of the tremendous
number, diversity and biological importance of non-coding (nc)RNAs. Because ncRNAs
typically function as ribonucleoprotein (RNP) complexes and not as naked RNAs,
understanding their biogenesis is crucial to comprehending their regulation and function.
The small nuclear and small nucleolar RNPs are two well studied classes of ncRNPs with
elaborate assembly and trafficking pathways that provide paradigms for understanding the
biogenesis of other ncRNPs.
Non-coding (nc)RNA
A functional RNA molecule
that does not code for a
protein (that is, it is not an
mRNA).
*Department of Genetics,
Case Western Reserve
University, Cleveland,
Ohio 44106-4955, USA.
‡
Departments of Biochemistry
& Molecular Biology,
and Genetics, University of
Georgia, Athens,
Georgia 30602, USA.
e-mails: [email protected];
[email protected];
[email protected]
doi:10.1038/nrm2124
Less than 2% of the human genome is translated into
protein, yet more than 40% of the genome is thought
to be transcribed into RNA1. The vast, untranslated
fraction of the human transcriptome includes a truly
remarkable number of functional non-coding (nc)RNAs2.
Indeed, the ongoing discovery of new classes of ncRNA
(for example, microRNAs, short interfering (si)RNAs,
repeat-associated RNAs and germline-specific RNAs)
and of new members of existing classes (for example,
small nucleolar (sno)RNAs) underscores the breadth and
depth of ncRNA function. Importantly, ncRNAs have
emerged as key trans-acting regulators of diverse cellular
activities in all three domains of life3–7 (TABLE 1). Among
the known activities of ncRNAs are: endonucleolytic
RNA cleavage and ligation, site-specific RNA modification, DNA methylation, DNA (telomere) synthesis and
modulation of protein function. These activities are
important (at many levels) for gene expression and also
for genome stability (TABLE 1).
In some cases, the molecular mechanisms by which
ncRNAs function are well understood, whereas in others
they are completely unknown. ncRNAs commonly function as adaptors that secure and position a nucleic-acid
target molecule for enzymatic activity that is catalysed
by an associated partner protein. Therefore, ncRNA
activity is typically driven by base pairing8 and often
involves several partner proteins; that is, the functional
unit is the non-coding ribonucleoprotein (ncRNP). The
steps of ncRNP assembly are often not well defined but,
importantly, can be regulated to control the activity of
the complex. Therefore, harnessing the tremendous
biomedical and applied research potential of ncRNPs
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(as both biomedical tools and therapeutic targets) will
require a detailed understanding of their biogenesis.
The small nuclear (sn)RNPs and snoRNPs are arguably the best studied examples of ncRNPs, and analysis
of their biogenesis has revealed an unanticipated complexity in their assembly, trafficking and mechanisms
of action. This review highlights significant aspects of
snRNP and snoRNP biogenesis that have emerged from
recent studies, placing them in the broader context of
earlier findings. The principles of RNP-complex formation and intracellular trafficking are probably not unique
to snRNPs and snoRNPs, and will probably also govern
the function of other ncRNPs. Therefore, we end with a
summary of the lessons learned from these RNPs that
might expedite our understanding of other ncRNPs.
The snRNAs
snRNAs comprise a small group of highly abundant,
non-polyadenylated, non-coding transcripts that function in the nucleoplasm. The snRNAs can be divided into
two classes on the basis of common sequence features
and protein cofactors. Sm-class RNAs are characterized
by a 5′-trimethylguanosine cap, a 3′ stem–loop and a
binding site for a group of seven Sm proteins (the Sm site)
that form a heteroheptameric ring structure (FIG. 1a).
Lsm-class RNAs contain a monomethylphosphate cap
and a 3′ stem–loop, terminating in a stretch of uridines
that form the binding site for a distinct heteroheptameric
ring of Lsm proteins (FIG. 1b).
The Sm class of snRNAs is comprised of U1, U2, U4,
U4atac, U5, U7, U11 and U12, whereas the Lsm class is
made up of U6 and U6atac. With the exception of the
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Table 1 | Functions of established and emerging non-coding RNAs
ncRNA
Function
RNA processing and modification
Spliceosomal snRNAs
Pre-mRNA intron splicing
U7 snRNA
Histone pre-mRNA 3′-end formation
C/D RNAs
2′ O-methylation of rRNAs, snRNAs and tRNAs;
rRNA processing
H/ACA RNAs
Pseudouridylation of rRNAs and snRNAs;
rRNA processing
RNase P RNA
pre-tRNA maturation (5′-end cleavage)
Trypanosomal guide RNAs
mRNA editing; production of alternative mRNAs
Transcription
7SK RNA
P-TEFb-mediated transcription-elongation control
6S RNA
Transcriptional regulation in bacteria by direct
interaction with RNA polymerase
Translation
tRNA
mRNA translation
tmRNA
Quality-control factor in bacteria; triggers release of
ribosomes stalled in translation
Protein trafficking
SRP RNA
Protein translocation to the endoplasmic reticulum
Regulation of gene expression
siRNAs
Gene silencing: cleavage of RNAs derived from
viruses, retroelements and repeat sequences
miRNAs
Gene silencing: translational repression or cleavage
of target mRNAs
piRNAs
Gene silencing: mammalian germline silencing of
repeat transcripts (chromatin modification?)
rasiRNAs
Gene silencing: Drosophila germline silencing of
repeat transcripts (chromatin modification?)
Eubacterial antisense RNAs
Gene silencing: translational repression or cleavage
of target mRNAs; in rare cases, gene activation is
observed
Genomic stability
Telomerase RNA
Telomere synthesis
miRNA, microRNA; ncRNA, non-coding RNA; piRNA, PIWI-interacting RNA; P-TEFb, positive
transcription elongation factor-b; rasiRNA, repeat-associated siRNA; RNase, ribonuclease;
rRNA, ribosomal RNA; siRNA, small interfering RNA; snRNA, small nuclear RNA; SRP, signal
recognition particle; tmRNA, transfer messenger RNA; tRNA, transfer RNA.
U7 snRNP, which functions in histone pre-mRNA 3′
processing, the other uridine-rich snRNPs form the
core of the spliceosome and catalyse the removal of
introns from pre-mRNA9. Accurate removal of intronic
sequences is guided by base-pairing interactions
between the spliceosomal snRNAs and the intron–exon
junctions. During the course of the splicing reaction,
specific and dynamic base-pairing interactions take
place among and between the snRNAs themselves.
More than 150 partner proteins are also involved
in this process, a subject that has been extensively
reviewed10,11.
Less well understood is the life history of the snRNPs
prior to their assembly into the spliceosome. The recent
studies highlighted here indicate that transcription and
3′ processing of most snRNAs are coupled by a system
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that is parallel to, but distinct from, that which produces mRNAs. Furthermore, the cellular systems that
function in nuclear transport and assembly of snRNPs
(and probably of other ncRNPs) will be described.
Coupled transcription and 3′ processing
The Sm-class snRNA genes share several common
features with protein-coding genes, including the arrangement of upstream and downstream control elements
(FIG. 1c). Sm-class genes are transcribed by a specialized
form of RNA polymerase II (Pol II) that is functionally
similar to the Pol II used by the mammalian proteincoding genes12. Promoter-swap experiments performed
more than two decades ago13,14 indicated that factors that
are important for accurate recognition of 3′-processing
signals (FIG. 1c) must load onto the polymerase at the
promoter; however, factors responsible for cleavage of
these non-polyadenylated transcripts have only recently
been identified.
Baillat et al.15 purified a complex from human cells,
called the Integrator, that associates with the C-terminal
domain (CTD) of Pol II and can be co-immunoprecipitated with snRNA promoter sequences, but not with
those of control mRNA or histone genes. Furthermore,
two of the purified proteins, INT9 and INT11, share
significant similarity with the cleavage and polyadenylation specificity factors CPSF-100 and CPSF-73,
respectively15, which are essential for the proper cleavage
and polyadenylation of the 3′ ends of mRNA and for
the formation of non-polyadenylated histone premRNA 3′ ends16,17 (FIG. 1c). Moreover, INT11 is probably
the endonuclease subunit of the Integrator complex, as
depletion of the endogenous protein or overexpression
of a construct containing a mutation in a conserved
metal-chelating residue inhibited 3′-end formation in
snRNA15. Therefore, it seems that CPSF-73 has a catalytic
role in two independent mRNA 3′-processing complexes
and that a related protein, INT11, has a similar role in
cleavage of Sm-class snRNA 3′ ends.
In metazoa, CPSF and Integrator complexes interact with the CTD of Pol II to couple transcription to
downstream RNA-processing events. The role of CPSF
in mediating communication between transcription
and polyadenylation is conserved among eukaryotes.
However, the putative role of the metazoan Integrator
proteins in coupling transcription and processing seems
to be carried out by the Nrd1 complex in yeast18–20.
Together with its partner proteins Nab3, Sen1 and others,
the Nrd1 complex binds to the Pol II CTD and directs
3′-end formation of non-polyadenylated transcripts such
as snRNAs and non-intronic snoRNAs21–23. The evolutionary relationship, if any, between the Nrd1-complex
proteins and those of the Integrator complex has not
been established.
The Lsm-class snRNA genes (U6 and U6atac) are
transcribed by Pol III using specialized external
promoters24. The run of uridines that forms the Lsmbinding site at the 3′ end (FIG. 1b) also doubles as a
Pol III transcription terminator. Therefore, there are
few parallels between Lsm-class genes and genes that
encode proteins.
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Nuclear export systems
In higher eukaryotes, Lsm-class snRNAs never leave
the nucleus, whereas the biogenesis of Sm-class
snRNPs is a multistep process that takes place in
distinct subcellular compartments. Sm-class snRNAs
are exported from the nucleus for cytoplasmic maturation events. The 5′ cap structure and length of the
RNAs are key determinants in nuclear export, and
the main components of the trafficking system have
been defined.
Following transcription and 3′ processing in the
nucleus, newly transcribed Sm-class snRNAs are
transported to the cytoplasm by an export complex
(FIG. 2) that contains the snRNA-specific export adaptor
protein PHAX, the export receptor chromosome region
maintenance-1 (CRM1; also known as exportin-1),
a Sm-class snRNA
b Lsm-class snRNA
CH3
CH3
CH3
GpppApNp
3′ stem
3′ stem
OCH3
MPG cap
Sm site
Lsm site
TMG cap
Consensus AAUUUUUGG
U7 snRNA AAUUUGUCU
E
F
D2
CH3
E
G
D1
UUUUU
pppGp
G
5
7
D3
F
D3
6
4
B
11
B
3
8
Sm core
10
U7 core
2
Lsm core
c Generic Pol II gene
Control region
Cleavage site
Coding region
Terminator
Processing signals
Control region
Adaptor protein
Bridges proteins that link
specific cargoes to their
cognate transport receptors.
Export/import receptor
Collectively called
karyopherins, these proteins
facilitate transport across
nuclear pores. Import factors
are called importins and export
factors are called exportins.
Receptors primarily mediate
interactions with nuclear pores
but can also bind cargo
directly.
Processing signals
Cleavage site
Gene product
Distal
Proximal
Upstream
Downstream
Site (factor)
snRNAs
DSE
PSE
3′ stem?
3′ box
?
Histones
CCPE
TATA box
HSL
HDE
CA (CPSF-73)
Proteins
Enhancer
TATA box
AAUAAA
G/U rich
CA (CPSF-73)
(INT11)
Figure 1 | Anatomical features of Sm- and Lsm-class small nuclear RNAs. a | Sm-class small nuclear (sn)RNAs are
transcribed by RNA polymerase II and contain three important recognition elements (boxed): a 5′-trimethylguanosine
(TMG) cap, an Sm-protein-binding site (Sm site) and a 3′ stem–loop structure. In metazoans, the Sm site and the 3′-stem
elements are required for recognition by the survival motor neuron (SMN) complex for assembly into stable core
ribonucleoproteins (RNPs), whereas the TMG cap and the assembled Sm core are required for recognition by the nuclear
import machinery. With the exception of U7, each of the Sm-class snRNPs contains a common core of seven Sm proteins
and a unique set of snRNP-specific proteins. The consensus sequence of the Sm site and the sequence of the specialized Sm
site of the U7 snRNA are shown. The consensus Sm site directs assembly of the canonical heptameric protein ring (Sm core).
In the U7 snRNP, two of the seven Sm proteins (SmD1 and SmD2) are replaced by LSM10 and LSM11, respectively, forming
the U7 core RNP. b | Lsm-class snRNAs are transcribed by RNA polymerase III and contain a 5′-monomethylphosphate (MPG)
cap, a 3′ stem and terminate in a stretch of uridine residues (the Lsm site) that is bound by the Lsm core. U6 and U6atac are the
only known Lsm-class snRNAs. c | Parallels in the organization of control elements for RNA polymerase II transcription and
processing of Sm-class snRNA genes, cell-cycle-dependent histone genes and canonical protein-coding genes. For each of
the three gene classes, the elements and factors involved in their expression are tabulated. The putative snRNA-gene
upstream-processing signal and the 3′-cleavage-site consensus sequence have yet to be determined. In addition to the
TATA box, control regions for the cell-cycle-dependent histone genes include an upstream cell-cycle-promoter element
(CCPE) and an internal element136 located in the protein-coding region (not shown). CPSF, cleavage and polyadenylation
specificity factors; DSE, distal sequence element; HSL, histone stem–loop; HDE, histone downstream element; INT11,
Integrator-11; PSE, proximal sequence element.
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F E G
Sm proteins
D3
D2
D1
B
Sm
core
SMN
complex
m7G
m7 G
CRM1
SMN
complex
Sm-core
assembly
CBC
RAN
GDP
Exo
Cap
hypermethylation
TGS1
3′-end
trimming
Sm
core
SMN
Imp-β complex
SPN TMG
Cytoplasm
NPC
snRNP
import
Nucleus
PHAX
SPN
Imp-β
IGC
Nucleolus
RAN CRM1
GTP
PHAX P
CBC m7G
snRNA export
IGC
TMG
PFs
Transcription
CB
?
7G
IGC
CB
Sm
core
SMN
complex
IGC
snRNP maturation
m
Figure 2 | Biogenesis of Sm-class small nuclear RNPs. The life-cycle of Sm-class small nuclear ribonucleoproteins
(snRNPs) includes both nuclear and cytoplasmic phases. Following transcription by a specialized form of RNA
polymerase II, pre-snRNA transcripts are exported to the cytoplasm, with a potential intermediate stop at Cajal bodies
(CBs). The snRNA-export complex consists of the export receptor chromosome region maintenance-1 (CRM1), the
hyperphosphorylated form of the export adaptor PHAX, the heterodimeric cap-binding complex (CBC) and the
GTP-bound form of Ran GTPase. These factors dissociate from the pre-snRNA in the cytoplasm after binding by the
survival of motor neuron (SMN) complex and dephosphorylation of PHAX. The SMN complex recognizes specific
sequence elements in the snRNAs (the Sm-protein-binding site and the 3′ stem–loop) and recruits a set of seven Sm
proteins, three of which contain symmetrical dimethylarginine residues (orange stars), to form the Sm-core RNP.
Following assembly of the Sm core, the 7-methylguanosine (m7G) cap is hypermethylated by trimethylguanosine
synthase-1 (TGS1) to form a 2,2,7-trimethylguanosine (TMG) cap structure, and the 3′ end is trimmed by a yet-to-beidentified exonuclease (Exo). The formation of the TMG cap triggers the assembly of the import complex, which
includes the import adaptor snurportin-1 (SPN) and the import receptor importin-β (Imp-β). Both SPN and the
SMN complex make functional contacts with Imp-β. On nuclear re-entry, the Sm-class snRNPs target to Cajal bodies
for snRNP maturation, which includes binding by snRNP-specific proteins and site-specific modification by small Cajal
body (sca)RNPs. Following discharge from Cajal bodies, the newly minted snRNPs either participate in splicing at
perichromatin fibrils (PFs) or are stored in interchromatin granule clusters (IGCs) for later use. NPC, nuclear pore
complex; P, phosphate.
Cajal bodies
Intranuclear structures that
function as ribonucleoproteinassembly, trafficking and
remodelling centres.
the cap-binding complex (CBC; which consists of the
protein heterodimer CBP80–CBP20) and Ran GTPase25.
The ability of PHAX to export snRNAs depends on its
phosphorylation status. Hyperphosphorylated PHAX is
predominantly nuclear. Together with CBP80 and CBP20,
PHAX forms a bridge between the snRNA and CRM1
(FIG. 2). Hypophosphorylated PHAX is predominantly
cytoplasmic, cannot bind to snRNAs and recycles to the
nucleus through the import receptor importin-β26. The
kinase(s) and phosphatase(s) that regulate this process
have not been identified. However, it is not clear whether
fungal snRNAs are exported, as their genomes do not
contain recognizable PHAX orthologues.
It is also not clear how PHAX distinguishes the 5′ caps
of certain snRNA precursors from those of bulk mRNAs.
A few studies have begun to shed light on this conundrum, although much work remains to be done. Ohno
and colleagues showed that when it comes to RNA export,
length really does matter27,28. By inserting increasingly
longer sequences at various positions in U1 snRNA, they
showed that export could be rerouted from the PHAX
pathway to the nuclear export factor-1 (NXF1)-mediated
mRNA-export pathway27,28. Conversely, intronless mRNAs
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can be progressively rerouted from the NXF1-mediated
to the PHAX-mediated pathway by successive deletions28.
Therefore, length is also an important determinant of
RNA-export-pathway choice.
However, RNA length and cap structure do not tell
the whole story, as several snoRNA transcripts are of
similar size and have the same 5′ caps as the snRNAs
yet are retained in the nucleus29,30. For example, recent
evidence indicates that PHAX binds to precursor U3
snoRNAs31 and targets them to Cajal bodies (rather than
to the cytoplasm), where they interact with the 5′-cap
hypermethylase trimethylguanosine synthase-1 (TGS1)
prior to their accumulation in nucleoli30. The current
view holds that assembly with core snoRNP proteins
(see below) and hypermethylation of the 5′ cap can
override snoRNP export. U3 transcripts with mutations
in conserved box C and D elements (FIG. 3) are exported
to the cytoplasm30,32,33. These results, together with the
findings of Smith and Lawrence34 indicating that U2
snRNA precursors that contain 3′ extensions also localize
in Cajal bodies, lead us to propose the existence of a
discrimination step for RNA export that takes place in and
around Cajal bodies.
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Cytoplasmic assembly of the core snRNP by SMN
On export to the cytoplasm, assembly into stable
Sm-core particles is carried out by the survival motor
neuron (SMN) protein complex (FIG. 2) . Evidence
indicates that the SMN complex also functions in the
assembly of other RNPs35,36. Loss-of-function mutations
a C/D
Target
RNA
in the human SMN1 gene cause a neurogenetic disorder
called spinal muscular atrophy (BOX 1). Together with its
associated factors, collectively known as Gemins, the
SMN complex binds to the newly exported snRNA precursors and to the seven Sm proteins that form the core
of the RNP35,37 (FIG. 2). Recent data point to a WD-repeat
b H/ACA
c U3 snoRNA (C/D)
CAB box
(Eukaryotes)
C/D
motif
(k-turn)
D′
Apical
loop
C′
C/D
motif
C
k-turn
(Archaea)
5 nt
Upper
stem
CH3
Guide
elements
CH3
5 nt
3′
5′
C
TMG
Guide
elements
(pseudoU
pocket) 5′
3′
3′
(H/ACA)
Target
RNA
Lower
stem
5′
D
d Telomerase RNA
3′
D
Terminal
stem
C/D
motif C′
Distance
15 nt
Nψ
C/D
motif
(k-turn)
B
5′
ACA
or
H
3′
CAB box
Ligated termini
(Archaea)
H
ACA
3′
TMG
Figure 3 | Anatomical features of C/D and H/ACA RNAs. a | The secondary structure of an archetypical
C/D 2′-O-methylation guide RNA. Conserved box C (RUGAUGA) and D (CUGA) sequence elements are tethered by the
terminal stem–loop and apical loop and form kink-turns (k-turns). A C/D pair is associated with an antisense element
(blue) located upstream of box D that base pairs with the target RNA (red). Target RNA is methylated on the ribose of the
nucleotide (nt) that is base paired with the guide RNA that is 5 nucleotides upstream of box D. b | Secondary structure of an
archetypical H/ACA pseudouridylation guide RNA. An imperfect hairpin is formed by a lower stem, pseudouridylation
(pseudoU) pocket, upper stem and an apical loop. The target RNA is bound within the pseudouridylation pocket. Box ACA
is found immediately downstream of the hairpin, ~15 nucleotides from the uridine that is modified (ψ; N represents a
conserved nucleotide that is located next to the target ψ). In eukaryotes, H/ACA RNAs consist of two hairpin units,
separated by a single-stranded hinge that contains box H (ANANNA). The apical loop can contain a Cajal body (CAB)-box
sequence element (consensus UGAG) for Cajal body retention in the subset of eukaryotic H/ACA RNAs that guide the
modification of small nuclear RNAs (rather than ribosomal (r)RNAs)115. In archaea, these RNAs contain a k-turn at the
junction of the upper stem and apical loop. c | U3 is a C/D RNA specialized to function in pre-rRNA processing. Antisense
elements involved in interacting with the target RNA are located in the 5′ half of the RNA. The RNA retains two C/D motifs
(boxed): a canonical C/D pair and a distinct B/C pair that is involved in interacting with U3-specific proteins. d | The 3′ half of
vertebrate telomerase RNA is a double hairpin H/ACA RNA (boxed), and the 5′ half is a pseudoknot required for telomere
synthesis. An antisense element that recognizes the DNA substrate is located in the pseudoknot domain. snoRNA, small
nucleolar RNA. TMG, 2,2,7-trimethylguanosine.
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Box 1 | Small RNPs and human disease
The study of small nuclear ribonucleoproteins (snRNPs) and small nucleolar (sno)RNPs
has contributed to our understanding of several important diseases.
Spinal muscular atrophy. Mutations in the survival motor neuron-1 (SMN1) gene result
in the degeneration of spinal motor neurons and severe muscle wasting37,132. The SMN
protein assembles Sm-class snRNPs, and probably also snoRNPs and other RNPs35,36.
Spinal muscular atrophy affects up to 1 in 6,000 people.
Dyskeratosis congenita. Mutations in key components of H/ACA RNPs are a cause of
dyskeratosis congenita, a rare syndrome that is characterized by abnormal changes in
the skin, nails and mucous membranes133. Long-term problems include bone-marrow
failure and cancer133. Apparently, the syndrome can arise from mutations in multiple
genes, including dyskerin (the orthologue of centromere binding factor-5 (Cbf5)),
telomerase RNA and telomerase reverse transcriptase. The involvement of proliferating
tissues is consistent with a primary defect in telomere maintenance.
Prader–Willi syndrome. This disorder affects as many as 1 in 12,000 people and is
characterized by extreme hunger, cognitive and behavioural problems, poor muscle tone
and short stature. The syndrome is clearly linked to the deletion of a region of paternal
chromosome 15 that is not expressed on the maternal chromosome. The deleted region
includes a brain-specific C/D RNA that seems to target the serotonin-2C receptor mRNA
and might regulate its editing66 or splicing67. Patients with Prader–Willi syndrome do not
express the C/D RNA and express reduced amounts of the functional serotonin-2C
receptor mRNA.
protein, Gemin-5, as the specificity factor that binds
snRNAs that contain a consensus Sm site38,39. It is currently unclear how the U7 snRNA, which contains an
unusual Sm site (FIG. 1a), is recognized by a subset of SMN
complexes that contain the U7-specific Sm-like proteins
LSM10 and LSM11 (REF. 40) . Additional factors in
the SMN complex help to bring the Sm proteins into the
fold, but the known mechanistic details are few and far
between.
Perichromatin fibril
(PF). A fine structure, which is
visible only under the electron
microscope, that is located
adjacent to transcriptionally
active chromatin and is
thought to be a site of active
pre-mRNA processing.
Interchromatin granule
cluster
(IGC).Corresponds to a nuclear
domain visible as a speckle
under the light microscope.
IGCs function as storage sites
for splicing factors that can be
recruited to perichromatin
fibrils.
Peptidyl transferase centre
Catalytic centre of the
ribosome that forms peptide
bonds during protein
translation.
mRNA-decoding centre
Region of the ribosome
involved in the selection of
transfer RNAs that correspond
to mRNA codons during
protein translation.
Import and assembly of snRNP-specific factors
Following the assembly of the Sm core, and in association with the SMN complex (FIG. 2), the snRNA 7-methylguanosine (m7G) cap is hypermethylated by the TGS1
protein to form a 2,2,7-trimethylguanosine (TMG) cap41.
In addition, a few nucleotides on the 3′ termini of the
snRNAs are trimmed away by a putative exonuclease,
apparently accelerating the import kinetics of snRNPs42.
Sm-class snRNPs contain two nuclear localization signals:
the TMG cap and the Sm core itself (FIGS 1,2). Each signal
uses the import receptor importin-β to transport snRNPs
into the nucleus43. However, the adaptor that specifically
recognizes the TMG cap is called Snurportin-1, whereas
the SMN complex (or a subcomplex thereof) is an adaptor for the Sm core44,45. The Sm-core and TMG-cap
import signals function independently in vitro 46,47,
however, it is likely that the adaptors (Snurportin-1 and
the SMN complex) function synergistically in vivo.
Once in the nucleoplasm, the snRNPs are free to
diffuse throughout the interchromatin space. Newly
made RNPs transiently accumulate in Cajal bodies (FIG. 2)
prior to accumulation in nuclear subdomains known as
perichromatin fibrils and interchromatin granule clusters48.
Additional RNP remodelling and assembly steps
are thought to take place in Cajal bodies35, including
RNA-guided modification of the spliceosomal snRNAs
(see below) and assembly of factors that are specific
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to a given species of snRNP 49–53. In addition to their
roles in de novo RNP assembly, Cajal bodies might also
be involved in recycling and remodelling U4/U6
snRNP complexes that are disrupted during the splicing
reaction49,52.
The snoRNAs
The RNAs commonly referred to as snoRNAs comprise
two families, the C/D and H/ACA RNAs. The term
small nucleolar RNA was originally coined to reflect the
nucleolar localization of the first members of this group
relative to their nucleoplasmic cousins, the snRNAs.
Most C/D and H/ACA RNAs function in ribosomal
(r)RNA modification and processing in the nucleolus.
However, the C/D and H/ACA RNAs have evolved an
impressive portfolio of functions and targets as well as a
corresponding range of cellular localization patterns that
includes sites outside of the nucleolus (to gain access to
different substrates). Therefore, as described here, the
snoRNA families have grown to include a diversity belied
by their increasingly anachronistic name. In addition,
we describe the roles of essential partner proteins in the
function of C/D and H/ACA RNAs, and the elaborate
pathways by which functional RNPs are assembled
and delivered.
Functional diversity and anatomy of the RNAs
Whereas eukaryotic cells contain fewer than a dozen
snRNA species, they contain more than 200 unique
C/D and H/ACA RNAs 54,55. The C/D and H/ACA
RNAs are among the most numerous and functionally diverse trans-acting ncRNAs currently known56–60.
Moreover, these RNAs are present in archaea as well as
in eukaryotes, indicating that they arose over 2–3 billion
years ago.
The C/D and H/ACA RNAs are essential for major
biological processes including protein translation,
mRNA splicing and genome stability (TABLE 1). Most
known C/D and H/ACA RNAs guide the modification of other ncRNAs. The two classes of RNA guide
different nucleotide modifications: C/D RNAs direct
2′-O-ribose methylation and H/ACA RNAs guide
pseudouridylation (conversion of uridine to pseudouridine). C/D and H/ACA RNAs function in parallel to
both process and modify ribosomal RNA. These RNPs
modify key regions of rRNA (for example, the peptidyl
transferase centre and the mRNA-decoding centre), and
both types of modification are essential for ribosome
function 60,61. Other modification targets include
snRNAs in eukaryotes62, transfer RNAs in archaea63,
spliced leader RNAs in trypanosomes64 and perhaps
at least one brain-specific mRNA in mammals65–67.
Spliceosome function also depends on the modification
of snRNAs by C/D and H/ACA RNAs60. In addition,
one H/ACA RNA, telomerase RNA, is required for telomere synthesis68. Moreover, the existence of substantial
numbers of orphan RNAs that do not apparently target
established substrates (that is, targets such as rRNAs
and snRNAs) indicates that C/D and H/ACA RNAs also
function on targets and in processes that remain to
be identified69,70.
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Box 2 | Composition and organization of C/D and H/ACA RNPs
a C/D RNP
b H/ACA RNP
L7Ae
C′
L7Ae D′
58
56/
p
No
Nop10
Fib
/58
Fib
p56
No
Gar1
Cbf5
D L7Ae
C
5′
C/D RNPs
ACA
3′
H/ACA RNPs
Archaea
Human
Archaea
Human
Fibrillarin
Fibrillarin
Cbf5
Dyskerin
L7Ae
15.5K/NHPX
L7Ae
NHP2
Nop56/58
NOP56
Gar1
GAR1
NOP58
Nop10
NOP10
The core archaeal C/D ribonucleoprotein (RNP) proteins are: fibrillarin (Fib; the
2′-O-methyltransferase), L7Ae and Nop56/58. Eukaryotes have homologous proteins.
The eukaryotic NOP56 and NOP58 are closely related paralogues that are both
associated with C/D RNAs (one with each C/D and C′/D′ unit134). These two proteins
have apparently evolved distinct, but crucial, functions in eukaryotes as both
are essential.
The core archaeal H/ACA RNP proteins are: centromere binding factor-5 (Cbf5;
the pseudouridine synthase), L7Ae, Gar1 and Nop10. Therefore, the L7Ae protein is a
common component of both C/D and H/ACA RNPs in archaea. In eukaryotes, distinct
L7Ae-related paralogues (for example, human 15.5K (also known as NHPX) and NHP2)
are essential components of the individual C/D and H/ACA RNPs.
In the archaeal C/D RNPs, fibrillarin acquires a guide RNA through Nop56/58 and
L7Ae (see part a of the figure). Cbf5 binds the guide RNA directly in the archaeal
H/ACA RNP (see part b in the figure). In both RNPs, L7Ae induces essential changes in
the conformation of the guide RNA.
Kink-turn (k-turn) motif
A common RNA structural
motif that is bound by a family
of related proteins, including
L7Ae, resulting in a sharp bend
(or kink) in the RNA helix.
C/D and H/ACA RNAs are defined by conserved
signature-sequence elements and characteristic
secondary structures (FIG. 3). The C/D and H/ACA
modification-guide RNAs are the archetypes, with
the simplest (and apparently most ancient) configurations. The RNAs recognize and secure target molecules
through antisense elements (FIG. 3) . The C/D and
H/ACA RNAs that are specialized to perform other
functions (including pre-rRNA processing and telomere synthesis) display variations on the basic architecture (FIG. 3). All of the RNAs of each family interact
with core sets of highly conserved proteins to form
the C/D and H/ACA RNPs (BOX 2). In the case of the
modification-guide RNPs, these proteins, along with
the guide RNA, are sufficient for function in vitro71–76.
Other proteins are necessary for specialized functions
and for function in vivo57,59.
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
Architecture of the C/D and H/ACA RNPs
Like other ncRNAs, the C/D and H/ACA modificationguide RNAs function as adaptors that link a catalytic
component of an RNP to a target. Reconstitutions of
functional archaeal C/D71–74 and H/ACA modificationguide RNPs75,76 have rapidly advanced our understanding
of the organization and function of these complexes.
Atomic-resolution structures of all of the core components of archaeal C/D 77–81 and H/ACA RNPs 82–86,
including several multicomponent complexes, are also
now available, providing spectacular structural insights.
The C/D and H/ACA RNPs illustrate different modes
for the recruitment and positioning of a partner enzyme
by a guide RNA, and a range of essential functions are
contributed by the other core proteins.
The H/ACA RNP: direct binding of the enzyme. The
simplest recruitment paradigm has been found in the
H/ACA RNP, in which the protein enzyme interacts
directly and specifically with the guide RNA through
conserved features of the RNA75,76,84,86. Centromere
binding factor-5 (Cbf5; known as dyskerin in humans)
is a pseudouridine synthase with a catalytic domain and
a PUA (pseudouridine and archeosine transglycosylase)
domain. Box ACA and the lower stem of the guide RNA
are bound by the PUA domain of Cbf5, anchoring the
antisense elements near the catalytic site86 (BOX 2). In
humans, point mutations that affect a cluster of amino
acids in the PUA domain of the dyskerin protein result
in dyskeratosis congenita82 (BOX 1). This dyskeratosis
congenita mutation cluster was uncovered by mapping
the widely dispersed dyskeratosis congenita mutations
in the context of the predicted three-dimensional structure of the human protein (modelled from the crystal
structure of archaeal Cbf5)82. The affected amino acids
are in the immediate vicinity of, but do not include,
those amino acids that are directly involved in guide
RNA–protein interactions (which presumably would
significantly disrupt RNA interactions and result in
more severe defects)82,86. Consistent with the hypothesis
that the disease-inducing mutations affect (but do not
eliminate) RNA binding, the amounts of H/ACA RNAs,
including telomerase RNA and snoRNAs, are reduced in
patients with dyskeratosis congenita and mouse models
of the disease87–89. Nonetheless, there are also other viable
hypotheses for the molecular basis of this disease82,86.
The other three H/ACA RNP proteins are nonetheless
essential for the function of the complex75. Gar1 and
Nop10 each bind to distinct sites on the catalytic domain
of Cbf5, whereas L7Ae interacts directly with the guide
RNA75,82–84,86 (BOX 2). Current evidence indicates that Gar1
is involved in the binding and/or release of the target
RNA82,86. Nop10 is positioned along the upper stem of the
guide RNA between Cbf5 and L7Ae, and appears to interact with both L7Ae and the guide RNA (as well as Cbf5) in
the context of the complex, indicating a coordinating function86. L7Ae binds the kink-turn (k-turn) motif that is located
in the upper stem–apical loop of the guide H/ACA RNA
(FIG. 3) and induces a major bend (or kink) in the RNA85,86.
It remains to be determined how the resulting changes
might contribute to the function of the H/ACA RNP.
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An H/ACA RNA can have one, two or three hairpins, and
the evidence indicates that each hairpin serves as a binding site for the complete set of four core H/ACA RNP proteins75,90. In mammals, H/ACA RNAs almost exclusively
consist of two hairpin units and the available evidence
indicates that the organization of the RNP is similar91,92.
a Cotranscriptional assembly
of inactive RNPs
5′
Nascent H/ACA or C/D RNP
snoRNA
CTD
Pol II
b Trafficking to Cajal bodies
and assembly of active
RNPs by factor exchange
snoRNA
Cajal body
c Targeting to functional
destination
Mature
H/ACA or
C/D RNP
Nucleolus
Cajal body
Telomere
?
Figure 4 | Coordinated synthesis, assembly and
trafficking of C/D and H/ACA RNPs. Current
information indicates an emerging model in which
major steps in the production of both C/D and H/ACA
ribonucleoproteins (RNPs) are coordinated.
a | Cotranscriptional assembly of inactive pre-RNPs (early
biogenesis). A subset of the core RNP proteins (red) and an
exchange factor (yellow) are associated with the
C-terminal domain (CTD) of RNA polymerase II (Pol II) at the
guide RNA transcription site, and assemble on the nascent
RNA, forming a metabolically stable, but inactive, pre-RNP.
b | Maturation to functional RNPs at Cajal bodies (late
biogenesis and functional activation). The pre-RNP moves
to Cajal bodies where final steps in the formation of
functional complexes can occur, including RNA
modifications and replacement of the exchange factor
with a missing core protein. c | Targeting to functional sites
(localization). Mature RNPs move to the sites where they
function, for example, to nucleoli, Cajal bodies or
telomeres. snoRNA, small nucleolar RNA.
216 | MARCH 2007 | VOLUME 8
The C/D RNP: a protein bridge to secure the enzyme.
The association of the methyltransferase fibrillarin with a
C/D guide RNA depends on a bridge formed by the other
two core proteins, L7Ae and Nop56/58 (REFS 71,73,74).
L7Ae nucleates the assembly of the C/D RNP. As in the
H/ACA RNP, L7Ae binds a k-turn motif in the C/D guide
RNA, in this case formed by the interaction of box C and
D elements (FIG. 3; BOX 2). The resultant restructuring
of the RNA seems to create a new binding site that is
recognized by the Nop56/58 protein93. Nop56/58 in turn
recruits the catalytic protein fibrillarin to complete the
assembly of the functional complex.
A given C/D RNA can have one or two functional
units (each comprised of box C, box D and an antisense
element; FIG. 3). Recent results also indicate protein
bridging between these functional units (BOX 2). Highresolution X-ray structures of fibrillarin and Nop56/58
reveal a fibrillarin–Nop56/58–Nop56/58–fibrillarin
complex in which two fibrillarin–Nop56/58 heterodimers are connected through extensive coiled-coil
interactions between the Nop56/58 proteins79. This
bridge complex has the potential to place Nop56/58 and
fibrillarin at the second functional unit, and explains
the apparent lack of requirement for L7Ae binding
at the second unit in some systems73,94. Moreover, a new
structure of the complex appears to capture a different
conformation, revealing a substantial hinge motion that
might be involved in the proper placement of fibrillarin
at the target modification sites (S. Oruganti, Y. Zhang,
H. Li, M. T., R. T., W. Yang and H. Li, unpublished
observations) and addressing a concern that arose from
the analysis of intracomplex distances that assumed no
movement79.
Regulated assembly and trafficking in eukaryotes
The results emerging from recent studies indicate that
the assembly and trafficking of C/D and H/ACA RNPs
are intricately regulated in eukaryotic cells (FIG. 4).
It seems that the complexes are assembled as inactive
pre-RNPs on nascent guide RNA transcripts at the
genes. The pre-RNPs are transported to Cajal bodies,
where evidence indicates that they are matured to functional complexes. Last, the RNPs must be forwarded to
the sites where they function, but in at least one case,
delivery seems to be regulated to control the activity of
the RNP. The regulation of transport and assembly seems
to involve transient interactions with various factors,
some of which are now known.
Co-transcriptional assembly of pre-RNPs. As an H/ACA
RNA is transcribed, three of the four core H/ACA RNP
proteins and an assembly factor, called nuclear assembly
factor-1 (Naf1), associate with the RNA (FIG. 4a). The
subset of three H/ACA RNP proteins — Cbf5, Nop10
and Nhp2 (an L7Ae homologue) — and Naf1 can
be observed at transcriptionally active H/ACA genes by
chromatin immunoprecipitation and other assays95–98.
Naf1 also interacts with components of the Pol II
transcriptional machinery, including the CTD of the
polymerase large subunit, indicating intimate coupling
of RNP assembly to transcription97,99.
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Box 3 | Genomic origins of C/D and H/ACA RNAs
Exon
Intronic
Polycistronic
Monocistronic
C/D and H/ACA RNAs are encoded in eukaryotic genomes in various ways, and
understanding this point has facilitated the discovery and study of the biogenesis of
these RNAs54,59,135.
Most mammalian and plant C/D and H/ACA RNA genes (shown as coloured arrows in
the figure) are located in the introns of genes that encode proteins involved in ribosome
biogenesis (top panel). Generation of functional C/D and H/ACA RNAs requires
processing of the introns, which are released following pre-mRNA splicing. Interestingly,
in some cases, the host genes do not appear to encode proteins; the intron-encoded
RNAs are the only obvious products of these genes.
Other C/D and H/ACA RNAs are derived from polycistronic transcripts that can contain
up to ten different RNAs (middle panel). In this case, each RNA is liberated from the
precursor RNA by endonucleolytic cleavages.
A few C/D and H/ACA RNAs are independently transcribed by RNA polymerase II
(bottom panel). Like mRNAs and small nuclear (sn)RNAs, the independently transcribed
C/D and H/ACA RNAs receive a monomethylated (m7G) 5′ cap, and like the snRNAs, the
cap is hypermethylated to the trimethylated (m2,2,7G) form.
TRAMP complex
A nuclear polyadenylation
complex consisting of Trf4
(a poly(A) polymerase), Air2
(a zinc-knuckle protein) and
Mtr4 (an RNA helicase).
TRAMP functions together with
the exosome as a qualitycontrol mechanism to
stimulate the degradation of
various aberrant target RNAs.
Exosome
A complex of 3′→5′
exonucleases that has
important roles in RNA
processing and turnover.
Naf1 might ensure the assembly of a stable H/ACA
pre-RNP that is inactive until Naf1 is exchanged for Gar1.
Evidence indicates that Naf1 and Gar1 interact with a common site on Cbf5 in a mutually exclusive manner 82,95,97,99.
Naf1 and Gar1 share a region of homology that probably
mediates binding to Cbf5, but the proteins are otherwise
substantially different. Gar1 is essential for the function
of H/ACA RNPs75, but is the only core protein that is
not required for the accumulation of H/ACA RNAs. Naf1
is required for the accumulation of all classes of H/ACA
RNA95,99–102. However, Naf1 is not found associated with
H/ACA RNPs that are functionally engaged. The exchange
of Naf1 for Gar1 could be a key step in the regulated
transition of pre-RNPs into active H/ACA RNPs.
The assembly of C/D RNPs also seems to occur
co-transcriptionally103–105 and to be tightly coupled to
pre-mRNA splicing106. The molecular link between these
two processes has been identified as a general splicing
factor, called IBP60, that binds upstream of intronic
C/D RNAs and has putative helicase activity, which seems
to trigger C/D RNP assembly107. C/D RNP assembly also
involves a potential exchange factor called Bcd1 (box C/D
RNA). Like Naf1, Bcd1 is essential for the accumulation
of all C/D RNAs108,109, but is not a stable component of
the mature RNPs. Bcd1 also seems to interact with the
Pol II machinery. As the only core protein that is not
required for the accumulation of C/D RNAs in eukaryotes,
Nop56 might be the Bcd1-exchange partner (analogous
to Gar1).
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
The C/D and H/ACA RNPs are also subject to
co-transcriptional quality control. Aberrant RNAs
or RNPs seem to be targeted for destruction by a
mechanism that involves nuclear polyadenylation by
the TRAMP complex and degradation by the exosome110.
This nuclear surveillance system seems to be closely
linked to transcription and 3′-end processing18,21,111,112.
There are several additional factors implicated in the
co-transcriptional assembly of C/D and H/ACA RNPs.
For example, two putative RNA or DNA helicases, called
RVB1 and RVB2 (also known as TIP49a and TIP49b, or
as p50 and p55), are essential for the accumulation of
both C/D and H/ACA RNAs31,113. These helicases might
have a role in chaperoning RNP assembly or in releasing
nascent transcripts from genes.
Maturation at Cajal bodies. All C/D and H/ACA RNPs
seem to be rapidly targeted to Cajal bodies114–116 where it
is thought that essential maturation steps occur (FIG. 4b).
As described above, PHAX might have a role in the
localization of certain C/D and H/ACA RNAs to Cajal
bodies. Cajal bodies are complex intranuclear structures
enriched in factors involved in the modification of RNA
and in the assembly of RNA–protein complexes35,117,118.
In particular, these factors include TGS1, the enzyme
responsible for 5′-cap hypermethylation of capped
C/D and H/ACA RNAs105, and the SMN complex. As
discussed above, the SMN complex is an established
snRNP-assembly factor37,119. However, the SMN complex also associates with both C/D and H/ACA RNPs.
Moreover, the SMN complex interacts with fibrillarin
and Gar1 through the same domains that mediate the
binding and assembly of snRNPs120–122, and the accumulation of U3 C/D RNA depends on the SMN complex31.
Distribution to functional sites. Depending on their
function, the C/D and H/ACA RNAs ultimately localize
to nucleoli, Cajal bodies or telomeres (FIG. 4c). Nucleolar
RNAs move quickly through Cajal bodies to nucleoli.
Nucleolar targeting of these RNAs depends on the signature sequences (box C and D, or box H and ACA)
and an adjacent stem that tethers the box elements123,124.
A subset of the C/D and H/ACA guide RNAs, called
small Cajal body (sca)RNAs62, remains in Cajal bodies
to modify snRNAs. Interestingly, the scaRNAs can be
hybrids that contain both C/D and H/ACA motifs62.
Retention of H/ACA scaRNAs in Cajal bodies is mediated by Cajal body (CAB) boxes (FIG. 3), however, the
H/ACA domain (that is, the nucleolar targeting element)
is also required for proper Cajal-body localization115.
Mutation of the CAB boxes results in the appearance
of the RNAs in nucleoli, indicating that the CAB signal
supersedes the nucleolar targeting signal115,125. A subset
of Sm proteins was recently shown to associate with the
scaRNAs in a CAB-box-dependent manner, and it will be
interesting to see whether the Sm proteins are important
for Cajal-body localization126. Recent evidence indicates
that PHAX and CRM1 interact with the few C/D RNAs that
are independently transcribed and m7G-capped (BOX 3),
and have a role in trafficking to Cajal bodies and nucleoli,
respectively 30,31. However, the vast majority of vertebrate
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REVIEWS
C/D and H/ACA RNAs are salvaged from introns and
remain uncapped (BOX 3), and the factors involved in
their trafficking remain to be identified.
An interesting regulatory paradigm has emerged
from recent studies of telomerase: an H/ACA RNP (in
vertebrates) that functions in telomere synthesis127,128.
These results indicate that access of telomerase RNA
(and its protein partner, telomerase reverse transcriptase
(TERT)) to its substrate is regulated as a function of the
cell cycle to restrict the activity of the telomerase enzyme
to S phase. In human cells, telomerase RNA is retained in
Cajal bodies throughout most of the cell cycle through
a CAB box127,128. At the same time, TERT is found in
distinct nuclear foci (that do not correspond to Cajal
bodies, telomeres or other known structures)127. During
S phase, both telomerase RNA and TERT move to telomeres127,128. The trafficking of both components seems
to involve intermediate steps in nucleoli and in foci that
arise immediately adjacent to Cajal bodies, indicating
further regulation in the biogenesis and transport of
this enzyme127,128. In yeast, telomerase resembles an
Sm snRNP129 rather than an H/ACA snoRNP, and therefore
trafficking of this enzyme is regulated by distinct pathways
in different organisms.
Paradigms for the biogenesis of ncRNPs
Over the past few decades, intensive study of snRNPs
and snoRNPs has rewarded us with an understanding
of RNA-mediated mechanisms of RNA modification,
cleavage and splicing. The concept of targeting enzymatic activity through guide RNAs that have antisense
elements (derived from studies of the snRNAs and
snoRNAs) has helped pave the way for the discovery
and understanding of many other ncRNAs.
From the snRNPs and snoRNPs, we have learned that
members of a given ncRNP family can function in distinct
processes. This can be accomplished by specializations in
the ncRNAs that result in interactions with different sets
of partner proteins (as in the case of the C/D and H/ACA
RNPs involved in rRNA processing). In some cases (for
example, as proposed for the spliceosome), enzymatic
activity might be intrinsic to the RNA.
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Acknowledgements
We apologize to our colleagues whose work has not been cited
or discussed in full, owing to space constraints. We thank current and past members of the Matera and Terns groups who
have contributed to the work in our laboratories. M.P.T. and
R.M.T. are grateful to C. Glover for continued scientific discourse. This work was supported by National Institutes of
Health (National Institute of Neurological Disorders and
Stroke and National Institute of General Medical Sciences)
and Muscular Dystrophy Association grants to A.G.M. and by
grants from the National Institutes of Health (National
Institute of General Medical Sciences and National Cancer
Institute) and the Nora L. Redman Fund to M.P.T. and R.M.T.
Competing interests statement
The authors declare no competing financial interests.
DATABASES
The following terms in this article are linked online to:
UniProtKB: http://ca.expasy.org/sprot
Cbf5 | CPSF-73 | CPSF-100 | dyskerin | Gar1 | INT11 | Nab3 |
Nop10 | Nrd1 | PHAX | Sen1
FURTHER INFORMATION
A. Gregory Matera’s homepage:
http://genetics.case.edu/faculty2.php?fac=gxm26
The Terns laboratory homepage:
http://www.bmb.uga.edu/rterns/index.html
Access to this links box is available online.
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