Cell proliferation strongly correlates with the rate of ribosome biogenesis and total
protein synthesis. The time for completion of one cell cycle is primarily determined by
the time needed for sufficient mass accumulation via protein synthesis.
If translation and proliferation rates are uncoupled, cells are not able to maintain cell
size and functions.
Cell growth requires prodigious numbers of ribosomes, the molecular factories that
carry out protein synthesis. Ribosome biogenesis thus underlies the cell´s capacity to
grow. (Lempiainen [Link] 2009)
The factors that control cell cycle progression include cyclins, as well as their associated
kinases and inhibitors.
Ribosome biogenesis to precede the proliferative response, to ensure that key
components of the cell-cycle control system are synthesizes only when the cell has
established a substantial capacity for protein synthesis in response to an external
stimulus. This may be particularly significant for cyclins, which have short half-lives, and
the mRNA of which are poorly recognized by the protein-synthesis apparatus because
of their highly structured 5´UTR.
As more ribosomes are generated and the translational capacity increases, the
pathway becomes desensitized, favoring the translation of other mRNAs. Thus,
ribosomal-protein mRNA first serves to increase protein-synthesis capacity and to
prevent the translation of mRNAs lacking a 5´TOP, such as cell cycle regulators, until an
appropriate pool of ribosomes has accumulated. (Thomas et. Al 2000)
The question is how is this ribosome biogenesis integrated with the proliferative
response in single cell cycle?
A study revealed that on mouse embryos lacking all interphase Cdks (2,3,4 and 6)
developed into midgestation by expressing only cdk1. Cdk1 binds to all cyclins,
resulting in the phosphorylation of the retinoblastoma protein (Rb) and the expression
of genes that are regulated by E2F transcription factors. (Santamaria [Link] 2007)
The decision to initiate a new cell cycle is primarily made during the G1 phase, which
includes two critical checkpoints. The first is the restriction (R) point, occurring around
3.5 hours after mitotic exit, where cells assess the presence and strength of mitogenic
signals. This checkpoint determines whether cells should commence another round of
division or enter a quiescent state (G0). The R point is characterized by the
accumulation of cyclin D; if cells lack sufficient growth factor signals, they exit the cell
cycle and enter G0.
The second checkpoint, known as the growth checkpoint, varies in duration across
different cell lines and depends on nutrient availability. This checkpoint is not sensitive
to growth factors but is regulated by the mammalian target of rapamycin (mTOR). In
this phase, cells evaluate their nutrient and energy status before committing to cell
growth and cytokinesis. Inhibition of mTOR leads to a late G1 arrest, occurring after
growth factor independence has been established, and is marked by the activation of
cyclin E-CDK2. (Foster et. Al 2011)
A defining feature of classical TOP motifs is the series of 4 to14 pyrimidines following
the +1C or U. (Phillippe et al., 2019)
All transcripts encoding ribosomal proteins, most translation factors, and some RNA-
binding proteins carry a cis-regulatory RNA element termed the 5’terminal
oligopyrimidine (5’TOP) motif. This motif corresponds to an invariant 5’-cytidine
followed by an uninterrupted tract of 4–14 pyrimidine nucleotides and preceded by the
7-methylguanosine triphosphate (m7Gppp) cap (Fonseca et al., 2014; Iadevaia et al.,
2008; Meyuhas and Kahan, 2015; Levy et al., 1991). This motif is essential for
translation regulation of TOP mRNAs (Biberman and Meyuhas, 1999; Avni et al., 1994;
Meyuhas and Kahan, 2015).
In response to provision of nutrients (such as amino acids, growth factors, glucose, or
oxygen), the growth-associated kinase complex mTORC1 boosts the production of
components of the translation machinery encoded by TOP mRNAs (Meyuhas and
Kahan, 2015; Chantranupong et al., 2015).
Upon mTORC1 inhibition, a cellular state that is associated with TOP mRNA translation
repression, it was demonstrated that LARP1 dissociates from mTORC1 and binds to the
5’TOP motif of TOP mRNAs (Fonseca et al., 2015). We have also shown that LARP1
associates with the 5’TOP motif via a LARP1 family-specific ‘DM15 region’ located
within its C-terminus (Lahr et al., 2015). In the present study, we confirm a direct
association between the DM15 region and the 5’TOP motif.
DM15 region of LARP1 specifically binds the 7-methylguanosine 5’5’ triphosphate
(m7Gppp) moiety and the invariant first cytidine of TOP mRNAs. Biochemical analyses
reveal that LARP1 selectively prevents the binding of eIF4E to the m7Gppp cap to block
the assembly of the eIF4F complex on TOP mRNAs.
The 5’TOP sequence binds to the highly conserved, positively charged surface of the
three tandem helix-turn-helix HEAT-like repeats of DM15, termed A, B, and C.
LARP1 plays a fundamental role in the specialized recognition of the m7GpppC motif, a
unique feature of TOP mRNAs that encode all the core protein components of the
ribosome and the translation apparatus. Cap-binding proteins display a variety of
mRNA regulatory functions (Topisirovic et al., 2011). LARP1 regulates translation and
stability of this class of mRNAs through specialized 5’TOP motif (Fonseca et al., 2015;
Lahr et al., 2015; Aoki et al., 2013) and cap recognition.
LARP1 functions as a repressor of TOP mRNA translation: overexpression of LARP1 in
mammalian cells leads to reduced TOP mRNA translation, as inferred by the
accumulation of TOP mRNAs in subpolysomal fractions. Conversely, depletion of LARP1
protein from mammalian cells leads to an accumulation TOP messages in heavy
polysomal fractions (Fonseca et al., 2015), indicating that LARP1 represses TOP mRNA
translation.
Poly(A) tail length of TOP mRNAs dynamically fluctuates in response to amino acid
availability. Under amino-acid-rich/mTOR active conditions, where mRNAs are actively
translated, TOP mRNA poly(A) tail length positively correlates with ribosome loading
onto the mRNA. The tail length gradually shortens over time, leading to the
accumulation of short-poly(A)-tailed TOP mRNAs. In contrast, under mTOR
inactive/AAS conditions, where translation is globally repressed, long-poly(A)-tailed
TOP mRNAs are in turn accumulated over time in a manner dependent on LARP1. We
show that LARP1 interacts with the non-canonical poly(A) polymerases PAPD4, PAPD5,
and PAPD7 and induces post-transcriptional polyadenylation of the target mRNAs when
tethered to the mRNA.
To investigate the correlation between poly(A) tail length and translation, we
performed polysome fractionation of cytoplasmic extracts and collected four fraction
pools corresponding to free (‘‘free’’), monosomal (‘‘mono’’), light-polysomal (‘‘light-
poly,’’ two to four bound ribosomes), and heavy-polysomal fractions (‘‘heavy-poly,’’
more than five bound ribosomes). Importantly, as 50 cap-purified RNAs were ligated
with a double-stranded adaptor with an oligo(dT)10 overhang that anneals to the 30 -
most end of a poly(A) tail, sequenced reads all originated from the intact non-
truncated transcripts. We first visualized bulk tail length distributions from all the reads
in each fraction and found that the density of longer poly(A)-tailed reads is slightly but
significantly higher in polysomal fractions (Figure 1B).
What might be the significance of the poly(A) tail elongation of TOP mRNAs during
AAS/mTOR inactivation? It obviously is not to activate translation during AAS, since
translation of TOP mRNAs is strongly repressed when mTOR activity is impaired
(Fonseca et al., 2015; Hong et al., 2017; Lahr et al., 2017; Philippe et al., 2018, 2020).
We assume that the long poly(A) tail could be a ‘‘triage’’ to ensure faster translational
resumption after the release from AAS. While translational repression of TOP mRNAs
occurs soon after mTOR inactivation (minutes), the tail elongation occurs hours later
(>4–8 h). Due to the long-term global translational shutdown and activation of
autophagy, the cellular protein levels are severely lowered, and translation factors are
in high demand right after release to immediately restore overall protein levels. Despite
the requirement of a certain level of translation machinery, the number of ribosomes is
also likely to be reduced, since mTOR inhibition triggers ribophagy (An and Harper,
2018).
Since the 5’TOP sequences are found in translation-related mRNAs, including all of the
ribosome subunits and other translation factors, TOP mRNAs must be translated with
the highest priority. (Ogami et al., 2022)
LARP1 has been previously shown to regulate the stability of TOP transcripts
(21,60,77). A recent study by (78) demonstrated that LARP1 exhibits poly(A) tail
lengthening activity – it is therefore tempting to speculate that may LARP1 bind to
PABP and the poly(A) tail of TOP mRNAs to protect these transcripts from
deadenylation. (Sonenberg et al., 2017)
LARPs 1 and 4 both exhibit 3ʹ poly(A) tail length protection-mRNA stabilization
associated with their La-modules and dependent on interactions with cytoplasmic
poly(A)-binding protein (PABP, aka PABPC1). For both, PABP-binding is mediated by the
PABP-interacting motif-2 (PAM2) adjacent to the Lamodule. PAM2 within the La-
module of LARP1 identified by Fonsesca et al.
Eukaryotic initiation factor eIF4G interacts via the backside of the β-sheet RNA-binding
surface of PABP RRM2, while the β-sheet is bound to the mRNA poly- (A) tail. In the
pre-initiation complex, eIF4G is bound to eIF4E, the 5ʹ m7 Gppp-cap binding protein
(Hinnebusch et al., 2017) a central regulatory point factor in health and disease. Thus,
PABP bridges the 5ʹ-end and the 3ʹ-poly(A) of an mRNA. (Mattijssen et al., 2020)
Central aspects of mRNA metabolism are determined by the length of the 3ʹ poly(A) tail
(PAT) and the cytoplasmic poly- (A) binding protein (PABP, PABPC1) that binds to it
[1,2]. PABP is a translation factor that also binds eukaryotic initiation factor eIF4G,
physically linking the mRNA 5ʹ initiation factors with the 3ʹ PAT.
Mutation of the PAM2 motif decreases LARP1 La-module activity for mRNA
stabilization and PABP binding. (Mattijssen et al., 2020)
LARP1 specifically recognizes the m7GpppC motif characteristic of TOP mRNAs. This
observation is particularly remarkable because the first cytdine is indispensable for
repression of TOP mRNA translation (Levy et al., 1991).
if DM15 recognizes the caps of TOP mRNAs, it might compete for cap binding with
eIF4E, the eukaryotic initiation factor required for canonical cap-dependent translation
initiation (Sonenberg et al., 1978, 1979)
DM15 from capped TOP RNA requires high-micromolar concentrations of eIF4E, low-
nanomolar concentrations of DM15 are sufficient to displace eIF4E from this substrate.
Interestingly, the opposite is true for capped non-TOP mRNA: eIF4E outcompetes
DM15 for the non-TOP substrate as the pre-bound or competitor protein (Figure 2—
figure supplement 3). These results are consistent with the preferred binding of eIF4E
for m7GpppG (Kiraga-Motoszko et al., 2011; Thoreen et al., 2012; et al., Tamarkin-Ben-
Harush et al., 2017)
most mRNAs have a purine in the +1 position (Schibler et al., 1977; Schibler and Perry,
1977), eIF4E is anticipated to stimulate their translation; by contrast, TOP mRNAs have
an invariant C in the first position, suggesting that LARP1 would sequester them,
thereby preventing cap-dependent translation initiation until a signaling event releases
this repression. Of note, we show that the affinity of the DM15 region of LARP1 for TOP
mRNAs is considerably higher than that of full-length eIF4E protein for this class of
mRNAs, likely explaining why excess eIF4E alone is insufficient to upregulate the
translation of TOP mRNAs (Shama et al., 1995).
Notably, the ability of TOP mRNAs to associate with eIF4G is markedly inhibited upon
overexpression of LARP1 in mammalian cells (Fonseca et al., 2015), consistent with the
idea that LARP1 competes with eIF4E and eIF4G for binding to TOP mRNAs.
Expression of wild type LARP1 hinders the binding of endogenous eIF4G to both RPS6
and RPL32 mRNAs, but not to b-actin mRNA.
LARP1 binds to the cap and 5’TOP sequence of TOP mRNAs, thus selectively inhibiting
eIF4F assembly on this class of transcripts. ([Link] et al., 2017)
LARP1 displaces eIF4E from capped TOP mRNAs, thereby preventing the assembly of
the eIF4F complex required for translation initiation.
whereby eIF4F stimulates TOP mRNA translation while LARP1 represses it. Notably,
LARP1 exhibits higher specificity and affinity for TOP mRNAs than eIF4E does,
suggesting it may function as the selective factor for TOP mRNA translation regulation.
1. IF4E: This protein binds to the 5' cap structure (m7G cap) of eukaryotic mRNAs.
It is a part of the eIF4F complex, which also includes eIF4G (a scaffolding
protein) and eIF4A (an RNA helicase).
2. PABP: PABP binds to the poly(A) tail at the 3' end of eukaryotic mRNAs. Its role
is crucial in stabilizing mRNA and enhancing translation efficiency.
3. Closed-Loop Formation: In the initiation of translation, PABP interacts with
eIF4G, which is part of the eIF4F complex. Since eIF4G is also associated with
eIF4E, this interaction effectively brings the 5' cap and the 3' tail of the mRNA
into close proximity, forming a 'closed-loop' structure. This structure is
important for efficient translation as it promotes ribosome recycling and
efficient scanning of the mRNA by the ribosome. (Mattijssen et al., 2021)
