Protein biosynthesis is one of the key elements of gene expression in living cells.
All proteins are synthesized on ribonucleoprotein complexes, the ribosomes. In bacteria
and their derivatives—eukaryotic mitochondria and plastids—ribosomes consist of a
small and a large subunit which together comprise more than 50 ribosomal proteins
and usually two to four ribosomal RNAs (rRNAs). Synthesis of the required rRNAs and
proteins, and their correct folding, maturation/modification and assembly into functional
particles are highly coordinated. However, whereas ribosomal composition and many
mechanistic aspects of their biogenesis are well understood, little is known about
the spatial organization of the procedure in bacteria and organelles. In eukaryotes,
the individual processes involved occur in defined regions of the cell: ribosomal
proteins are synthesized in the cytosol, but most rRNAs are transcribed, processed
and modified in the nucleolus, a distinct subnuclear compartment (Lafontaine and Tollervey,
2001; Boisvert et al., 2007). Only the transcription of the small 5S rRNA occurs in
the nucleoplasm. After their synthesis, ribosomal proteins and assembly factors are
imported into the nucleus, where they are combined with the appropriate rRNAs. Subsequently,
small and large ribosomal subunits are exported into the cytosol, where they pair
up to form functional ribosomes.
This Opinion Article focuses on recent findings which support the idea that, as in
eukaryotes, ribosomal biogenesis in bacteria, mitochondria, and plastids is spatially
organized. In these systems, there is growing evidence that rRNA processing and ribosome
assembly most likely take place in association with the nucleoid.
rRNA processing, maturation and ribosome assembly in bacteria
The processes of rRNA maturation and ribosome assembly are probably best understood
in bacteria (reviewed in Kaczanowska and Rydén-Aulin, 2007; Shajani et al., 2011).
In E. coli, the small 30S ribosomal subunit contains 21 ribosomal proteins and a 16S
rRNA, while the large 50S subunit consists of 33 proteins and two rRNAs, the 23S and
5S rRNAs (reviewed in Melnikov et al., 2012). All rRNAs are encoded in a polycistronic
gene cluster and transcribed as a single precursor, which undergoes extensive processing
and maturation to generate the mature rRNAs (reviewed in Deutscher, 2009; Shajani
et al., 2011). The processing reactions are carried out by the exo- or endonucleolytic
activities of at least five ribonucleases (RNases), including RNases III, E, G, T,
and YbeY (reviewed in Deutscher, 2009; Davies et al., 2010). Furthermore, the 23S
and 16S rRNAs undergo methylations and pseudouridylations at several positions, which
are assumed to influence ribosomal structure and function (reviewed in Shajani et
al., 2011).
Many of these maturation steps are coupled and take place on nascent rRNAs. The final
rRNA folding pattern is probably determined by an ordered sequence of interactions
with ribosomal proteins and assembly factors, which induce conformational changes
and stabilize the proper rRNA structures (reviewed in Shajani et al., 2011).
It was long believed that DNA-associated bacterial ribosomes translate nascent mRNAs
while these are being synthesized by the RNA polymerase (co-transcriptional translation).
Striking evidence for this hypothesis came from early electron microscopy studies
showing ribosome arrays (polysomes) attached to mRNA strands that were being transcribed
from DNA by multiple RNA polymerase (RNAP) molecules (Miller et al., 1970). However,
recent research suggests that, at least in E. coli and B. subtilis, most translation
is not coupled to transcription, and that cotranscriptional translation may be limited
to mRNAs encoding membrane proteins. By tracking the distribution of RNA polymerases
(as markers for the nucleoid DNA) and ribosomes by means of fluorescence microscopy,
a clear segregation of the nucleoid from ribosome-rich regions of the cytoplasm was
observed (Lewis et al., 2000; Bakshi et al., 2012; Chai et al., 2014). Bakshi et al.
(2012) found approximately 85% of the ribosomes in the ribosome-rich regions, while
only 10 to 15% were detected in close proximity to the nucleoid. The majority fraction
probably comprises actively translating “protein factories.” The nucleoid-associated
particles are thought to be in various stages of assembly, as several rRNA maturation
steps occur in a cotranscriptional and assembly-assisted manner (reviewed in Shajani
et al., 2011).
Further data support the identification of the nucleoid as the site of early rRNA
processing events. RNase III, which cotranscriptionally cleaves the primary rRNA transcript
to yield 16S, 23S, and 5S precursors, has been shown to be required for localization
of the pre-16S rRNA 5′ leader region to the nucleoid (Malagon, 2013). In RNA-FISH
assays for single-cell visualization, Malagon (2013) found that the nucleoid localization
of the 16S 5′ leader was dependent on the catalytic activity of RNase III, which provides
indirect evidence for the presence of the enzyme in this region. Based on evidence
for interplay between the Nus transcription elongation factors and RNase III in the
modulation of pre-rRNA biogenesis (Bubunenko et al., 2013), Malagon further speculated
that Nus proteins might serve to localize pre-rRNAs to the nucleoid.
Localized rRNA processing and ribosome assembly in organelles
Eukaryotic plastids and mitochondria are descended from endosymbiotically acquired
bacteria, and have retained their own genomes and machineries for gene expression
during evolution. It is therefore not surprising that many aspects of these genetic
systems, including genome organization and ribosome structure, resemble those of bacterial
rather than eukaryotic systems. Similarly, evidence for sublocalization of rRNA processing
and ribosome assembly events to the organellar nucleoids is now emerging.
Mitochondria
Evidence for rRNA maturation and ribosome assembly in association with the mitochondrial
nucleoid mainly derives from studies on mammalian mitochondria. Affinity purification
of established protein components of the mitochondrial nucleoid results in copurification
of ribosomal proteins and the ribosome assembly factor ERAL1 (He et al., 2012b). ERAL1,
a GTP-binding protein with RNA-binding activity, which had previously been linked
with a number of nucleoid proteins, interacts both with proteins of the small mitoribosomal
subunit and its rRNA component (Dennerlein et al., 2010; Uchiumi et al., 2010). ERAL1
was proposed to act as a chaperone for the small ribosomal RNA, as its depletion leads
to destabilization of the small subunit (Dennerlein et al., 2010).
Similarly, another mitoribosome assembly factor, the human GTPase NOA1 (C4orf14),
a homolog of the plant Rif1 protein, has been found together with the small ribosomal
subunit and translation factors in affinity-purified nucleoids (Flores-Pérez et al.,
2008; He et al., 2012a). This finding prompted the suggestion that assembly of the
small subunit in the mitochondrial nucleoid enables the direct transfer of newly transcribed
mRNAs to the ribosome.
Like bacterial rRNAs, mammalian mitochondrial rRNAs undergo site-specific methylations,
although to a lesser extent (reviewed in Rorbach and Minczuk, 2012). The enzymes responsible
belong to a family of rRNA methyltransferases, some of whose members have been reported
to modify nascent rRNAs in association with the nucleoid (Lee et al., 2013). A more
detailed analysis of one of these methyltransferases (named RNMTL1) revealed an additional
interaction between this protein and the large ribosomal subunit, suggesting that
assembly of mitochondrial ribosomes begins before rRNA transcription is complete (Lee
et al., 2013).
Moreover, a very recent study by Bogenhagen et al. (2014) provides evidence that mammalian
mitochondrial RNA processing enzymes, like RNase P and ELAC2 (tRNaseZL), as well as
a number of nascent mitochondrial ribosomal proteins associate with nucleoids to initiate
RNA processing and ribosome assembly. The authors therefore propose the mtDNA nucleoid
as a critical control center for mitochondrial biogenesis.
Plastids
An extensive body of evidence for localized ribosome biogenesis comes from plastids.
Besides numerous ribosomal proteins, the majority of proteins with known roles in
ribosome biogenesis have been identified in a comprehensive proteomic analysis of
the maize chloroplast nucleoid (Majeran et al., 2012; reviewed in Germain et al.,
2013). Many of these nucleoid-enriched ribosome biogenesis factors function in rRNA
processing, maturation and modification, as well as in ribosome assembly (summarized
in Table 1).
Table 1
Nucleoid-localized proteins with proposed functions in rRNA processing, maturation
and ribosome assembly in plant chloroplasts.
Protein
Protein family/domain
Proposed function in ribosome biogenesis
Maize identifier
Arabidopsis identifier
References
a
PROCESSING/MATURATION OF RRNA PRECURSOR TRANSCRIPTS
BPG2
YqeH -GTPase
Pre-rRNA processing
GRMZM2G035042_P01
AT3G57180
Komatsu et al., 2010; Kim et al., 2012
CSP41B
Endonuclease; Rossmann fold Epimerase/dehydrogenase
Processing of 23S 1st hidden break
GRMZM2G080546_P02
AT1G09340
Beligni and Mayfield, 2008; Bollenbach et al., 2009; Qi et al., 2012
PNPase
3′ to 5′ exonuclease
23S rRNA 3′ end maturation
GRMZM2G377761_P01
AT3G03710
Walter et al., 2002
PRBP
Similarity to DEAD box helicases
Processing of 4.5S precursors
GRMZM2G436328_P01
AT2G37920
Park et al., 2011
RAP
*
Octotricopeptide repeat (OPR) protein, RNA binding
Processing of 16S rRNA precursors
GRMZM2G050845_P01
AT2G31890
Kleinknecht et al., 2014
RBF1
Ribosome-binding factor A family protein
Processing of 16S rRNA precursors
GRMZM2G115156_P01
AT4G34730
Fristedt et al., 2014
RH39
DEAD box RNA helicase
Processing of 23S 2nd hidden break
GRMZM2G175867_P02
AT4G09730
Nishimura et al., 2010
RHON1
Rho transcription termination domain
Processing of 23S-4.5S precursors
GRMZM2G168335_P01
AT1G06190
Stoppel et al., 2012
RimM
RimM family
16S processing, ribosome assembly (in prokaryotes)
GRMZM2G165694_P01
AT5G46420
Bylund et al., 1998; Guo et al., 2013
RNase E
Endonuclease
Processing of 23S-4.5S precursor
GRMZM2G328309_P01
AT2G04270
Stoppel et al., 2012
RNase J
5′ to 3′ exonuclease and endonuclease
5′ end maturation of 16S and 23S rRNA precursors
GRMZM2G103315_P01
AT5G63420
Bollenbach et al., 2005; Sharwood et al., 2011
RNase P
*
(PRORP1/2)
Endonuclease
5′ end maturation of tRNA precursors
GRMZM2G002642_P01
AT2G32230
Gutmann et al., 2012; Bogenhagen et al., 2014
GRMZM2G418206_P01
RNase Z
*
Endonuclease
3′ end maturation of tRNA precursors
GRMZM2G125844_P01
AT2G04530
Canino et al., 2009; Bogenhagen et al., 2014
SVR1
Pseudouridine synthase
rRNA processing
GRMZM2G315806_P01
AT2G39140
Yu et al., 2008
rRNA MODIFICATION/RIBOSOME ASSEMBLY
DER
*
(EngA)
GTPase
rRNA processing/ ribosome biogenesis
GRMZM2G302233_P01
AT3G12080
Jeon et al., 2014
ERA
GTPase
Assembly of small ribosomal subunit (in prokaryotes and mitochondria)
GRMZM2G158024_P02
AT5G66470
Dennerlein et al., 2010; Uchiumi et al., 2010; He et al., 2012b
ObgC
ObgC - GTPase
Ribosome assembly
GRMZM2G077632_P01
AT5G18570
Bang et al., 2012
PFC1
rRNA methylase
16S rRNA methylation
GRMZM2G174669_P01
AT1G01860
Tokuhisa et al., 1998
RH22
DEAD RNA box helicase
Assembly of 50S ribosomal subunit
GRMZM2G100043_P01
AT1G59990
Chi et al., 2012
RH3
*
DEAD box RNA helicase
Assembly of 50S ribosomal subunit, group II intron splicing
GRMZM2G163072_P01
AT5G26742
Asakura et al., 2012
GRMZM2G415491_P01
RIF1
*
(NOS1, NOA1)
GTPase (YqeH)
Ribosome assembly
GRMZM2G384293_P01
AT3G47450
Flores-Pérez et al., 2008; Liu et al., 2010
RsmD
rRNA methylase
16S rRNA methylation (in prokaryotes)
GRMZM2G010801_P01
AT3G28460
Lesnyak et al., 2007
All proteins listed were identified as nucleoid proteins by Majeran et al. (2012).
Proteins known to be involved in ribosome biogenesis but not identified in their nucleoid
fraction, as well as factors that probably have only indirect effects on rRNA maturation
and ribosome assembly, are not included in this list.
*
Nucleoid localization demonstrated by groups other than (Majeran et al., 2012) (see
text).
a
Only most relevant publications referring to a function in ribosome biogenesis or
nucleoid localization are listed.
Among the processing and splicing factors identified are many plant homologs of bacterial
RNases that have been reported to be responsible for exo- and endonucleolytic cleavage
of the large rRNA precursor and maturation of cotranscribed tRNAs (reviewed in Stoppel
and Meurer, 2012; Germain et al., 2013). Most of the nucleoid-enriched proteins involved
in ribosome assembly and rRNA modification are classified as either GTPases, RNA helicases
or rRNA methylases (Table 1).
Two of the nucleoid-enriched plastid proteins listed in Table 1 are homologous to
the mitochondrial enzymes ERAL1 and NOA1, for which a nucleoid localization has also
been reported (see above). For several others, links with the nucleoid are further
supported by alternative approaches as exemplified in the following.
An immunological analysis of maize chloroplast subfractions revealed that the DEAD-box
RNA helicase RH3, thought to function in assembly of the 50S subunit, localizes to
the chloroplast stroma and thylakoids, as well as to nucleoids (Asakura et al., 2012).
For two recently characterized plant proteins required for ribosome biogenesis, DER
and RAP, cytological evidence for nucleoid localization comes from GFP fusion experiments
(Jeon et al., 2014; Kleinknecht et al., 2014). DER is a Double Era-like GTPase, whose
bacterial homolog (also known as EngA) acts as a ribosome assembly factor in E. coli,
was found to bind to the 50S ribosomal subunit and to play a role in pre-rRNA processing
in tobacco (Hwang and Inouye, 2006; Jeon et al., 2014). The Arabidopsis protein RAP
is a member of the Octotricopeptide Repeat (OPR) protein family, and binds to the
5′ leader sequence of the 16S rRNA precursor (Kleinknecht et al., 2014). Depletion
of RAP specifically affected the trimming/processing of the chloroplast 16S rRNA precursor,
which supports the identification of the nucleoid as the site of 16S rRNA processing
in chloroplasts. Preliminary data imply that RAP has no intrinsic RNase activity,
and might influence 16S rRNA maturation by conferring sequence specificity on an RNase
or by modulating RNA secondary structures to control the accessibility of an RNase
recognition site within the 16S precursor (Kleinknecht et al., unpublished results).
However, in the case of many chloroplast proteins reported to be involved in various
steps of ribosome biogenesis, GFP-based fusion studies provide no support for specific
localization to the plastid nucleoid. One possible reason for this may be the use
of transit peptide-GFP instead of full-length protein fusions, as determinants of
nucleoid localization are unlikely to be encoded in the transit peptide. Accordingly,
Park et al. (2011) also reported the localization of the protein PRBP, which likely
functions in 4.5S rRNA processing, to distinct spots within chloroplasts, which probably
represent nucleoids. This sublocalization was only observed when the full-length protein
was fused to GFP and not with a transit peptide-GFP fusion. Nevertheless, other proteins
for which full length-GFP fusions were used could not be clearly assigned to the nucleoid
(e.g., Flores-Pérez et al., 2008; Yu et al., 2008; Chi et al., 2012). This might be
due to weak or transient association of the respective protein with the nucleoid.
Alternatively, some of these proteins may perform further functions in other organellar
subcompartments leading to ambiguous localization signals.
Conclusion and future perspectives
Localization of rRNA processing and ribosome assembly to organellar nucleoids seems
to be a general phenomenon derived from a bacterial ancestor. Unlike the case of the
eukaryotic nucleus, no physical barrier intervenes between bacterial/organellar RNA
synthesis and translation. The nucleoid might therefore provide a scaffold for an
intra-organellar microenvironment which enables coupling of rRNA transcription to
ribosome assembly. This might not only enhance the efficiency of ribosome assembly
by substrate channeling, but also largely prevent the precocious association of mRNAs
with immature 30S ribosomal subunits. However, it remains to be shown whether the
colocalization of rRNA processing and ribosome assembly with nucleoids has functional
significance or simply reflects the rapid kinetics with which ribosome biogenesis
factors bind to nascent rRNA targets.
Nonetheless, the growing awareness of the requirement for sublocalized cellular processes
in apparently less organized bacteria and organelles, and the availability of more
sensitive detection techniques including super-resolution imaging, will undoubtedly
lead to new insights into the spatiotemporal organization of ribosome biogenesis in
the future.
Conflict of interest statement
The author declares that the research was conducted in the absence of any commercial
or financial relationships that could be construed as a potential conflict of interest.