ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 1, pp. 32-43 © Pleiades Publishing, Ltd., 2025.
Published in Russian in Biokhimiya, 2025, Vol. 90, No. 1, pp. 35-47.
32
MINI-REVIEW
A Portrait of Three Mammalian
Bicistronic mRNA Transcripts, Derived
from the Genes ASNSD1, SLC35A4, and MIEF1
Dmitry E. Andreev
1,2,a
* and Ivan N. Shatsky
2,b
1
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia
2
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia
a
e-mail: cycloheximide@yandex.ru
b
e-mail: ivanshatskyster@gmail.com
Received October 4, 2024
Revised November 25, 2024
Accepted December 7, 2024
Abstract— Recent advances in functional genomics have allowed identification of thousands of translated short
open reading frames (sORFs) in the 5′ leaders of mammalian mRNA transcripts. While most sORFs are unlikely
to encode functional proteins, a small number have been shown to have evolved as protein-coding genes. As a
result, dozens of these sORFs have already been annotated as protein-coding ORFs. mRNAs that contain both
a protein-coding sORF and an annotated coding sequence (CDS) are referred to as bicistronic transcripts. In
this study, we focus on three genes – ASNSD1, SLC35A4, and MIEF1 – which give rise to bicistronic mRNAs.
We discuss recent findings regarding functional investigation of the corresponding polypeptide products, as
well as how their translation is regulated, and how this unusual genetic arrangement may have evolved.
DOI: 10.1134/S0006297924603630
Keywords: translation initiation, reinitiation, leaky scanning, dual coding, bicistronic mRNA
Abbreviations: CDS, coding sequence; ISR, integrated stress response; sORF, short open reading frame; PFDL, prefoldin-
like module; RNP, ribonucleoprotein particle; uORF, upstream open reading frame
* To whom correspondence should be addressed.
INTRODUCTION
Pioneering works by Marylin Kozak has allowed
us to understand the basic mechanism of translation
initiation in eukaryotic cells, which is based on the
ribosomal scanning through the mRNA 5′-leader se-
quence [1]. According to this scanning model, the m
7
G
cap at the 5′-end of all mRNA molecules is first rec-
ognized by the eIF4F, which allows the 43S ribosom-
al complex recruitment on mRNA via the eIF4F-eIF3
interaction. The 43S complex then begins to scan the
5′-leader sequence in the 5′ to 3′ direction until the
start codon (usually AUG) is recognized. This trig-
gers detachment of the initiation factors, association
with the 60S subunit, and allows elongation process
to begin (see reviews [2-5]).
The mRNA 5′-leader sequences play an essential
role in the scanning mechanism, as the obstacles along
the path of the scanning ribosome complex decrease
the number of ribosomes that successfully reach the
AUG start codon of the main open reading frame.
Interestingly, approximately half of the mammalian
mRNAs contain at least one upstream AUG codon that
creates an upstream reading frame (uORF) [6-10]. With
the development of ribosome profiling, which allows
detecting translating ribosomes with single-nucleotide
resolution genome wide [11], it has been possible to
demonstrate widespread translation events within the
5′-leaders, thus making the accepted terminology of
5′-untranslated regions (5′-UTRs) obsolete.
Functional roles of uORFs are under intense in-
vestigation. A growing number of studies have shown
that some uORFs are involved in control of translation
of the main open reading frame (ORF), but we will
not focus on this aspect as it has been extensively
covered in several recent reviews [4, 12-21]. In addi-
tion to their regulatory functions, some uORFs also
encode functional polypeptides. Bicistronic mRNAs
BICISTRONIC mRNAs IN MAMMALS 33
BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025
are a type of mRNAs that encode two proteins in two
separate ORFs, and the ratio of these proteins can be
regulated at the translation level. Here, we will dis-
cuss three examples of mammalian bicistronic mRNAs
that have been extensively studied, and speculate how
their translation can be regulated. We will also pro-
pose how changing the ratio of the two polypeptides
for these selected cases can affect cell physiology.
ASDURF-ASNSD1
The ASNSD1 gene locus encodes a poorly charac-
terized protein that is predicted to have asparagine
synthase (glutamine-hydrolyzing) activity. Additional-
ly, there is a short protein coding gene called ASDURF
(ENSG00000286053), which is either expressed as a
separate ORF due to utilization of an alternative polya-
denylation site located in exon 4 (within the ASNSD1
CDS [coding sequence]), or as an upstream ORF in an
alternative transcript. According to the cap analysis of
gene expression (CAGE) data from the FANTOM5 proj-
ect [22] (visualized in the Zenbu browser [23]), these
two genes share a single promoter. Therefore, ASNSD1
could only be translated from the same mRNA, which
also contains the upstream ASDURF (Fig. 1a, Fig. S1A
in the Online Resource 1).
ASDURF (a.k.a. ASNSD1-SEP) was initially iden-
tified by Slavoff et al. [24] by using peptidomics
approach optimized for identification of the short
ORF-encoded peptides. Using isotope dilution mass
spectrometry [25], it was demonstrated that the intra-
cellular concentration of ASDURF in K562 cells was
386 molecules per cell [24]. The function of ASDURF
remained enigmatic until 2020, when Cloutier et al.
provided compelling evidence that it was a subunit
of the prefoldin-like module (PFDL) of PAQosome [26].
The PAQosome or Rvb1–Rvb2–Tah1–Pih1/prefoldin-like
(R2TP/PFDL) complex is a specific chaperone complex
responsible for assembly and maturation of many
key multiprotein complexes in mammalian cells [27].
In mammals, this complex consists of two modules,
R2TP and PFDL. Cloutier et al. first implemented the
proximity-dependent biotinylation (BioID) to identify
new interacting proteins with two known PAQosome
subunits, PIH1D1 and UXT, and found that the endog-
enous ASDURF was highly enriched in both experi-
ments. Next, the FLAG-based affinity purification mass
spectrometry (AP-MS) was also performed on two ad-
ditional PAQosome subunits, RPAP3 and URI1. Again,
ASDURF was among the highly enriched interacting
proteins. Furthermore, the FLAG-based purification
of ASDURF revealed subunits of the PAQosome, con-
firming that ASDURF is an integral component of the
PFDL module. In vitro assembly of the PFDL complex
from the purified components showed that ASDURF
is the 6th subunit of PFDL heterohexamer complex,
which also consists of UXT1, PFDN6, PDRG1, URI1, and
PFDN2. Interaction of ASDURF with the PFDN subunits
has also been independently confirmed by Hofman
et al. [28].
PAQosome is involved in the activity of many
multi-subunit protein and ribonucleoprotein particle
(RNP) complexes such as ribonucleoproteins of the
L7Ae family (box C/D and H/ACA snoRNPs, U4 snRNPs,
and telomerase and selenoprotein mRNPs), U5 snRNP,
phosphatidylinositol 3-kinase-related kinase complex-
es (PIKKs) (ATM, ATR, DNA-PKcs, mTOR, SMG-1, and
TRRAP), and RNA polymerases I, II, and III [33-38].
If ASDURF is essential for the PAQosome function,
its absence could affect cell fate. Recently, using the
CRISPR-Cas9 screens, Hofman et al. [28] demonstrated
that targeting ASDURF decreases cell viability in the
MYC-driven medulloblastoma cell lines. Importantly,
re-expression of ASDURF rescues this phenotype.
In vivo, the knockout of ASDURF prolonged the over-
all survival of mice with orthotopic xenografts of the
D425 MYC-driven medulloblastoma cells. Interestingly,
the ASDURF knockout in vitro reduced S-phase incor-
poration of bromouridine, suggesting that ASDURF/
PFDL activity could be involved in the cell cycle
control in the MYC-driven medulloblastoma. Less is
known about the ASNSD1, which is encoded by the
second cistron. ASNSD1 has an N-terminal class-II glu-
tamine amidotransferase domain and a C-terminal as-
paragine synthase B domain belonging to the adenine
nucleotide alpha hydrolase (AANH) superfamily, and
it is likely involved in asparagine synthesis.
Analysis of the aggregated riboseq datasets from
human cells reveals three translated regions: a short
uORF encoding the MP dipeptide located 46 nts up-
stream of the first cistron, the ASDURF (first cistron),
and the ASNSD1 (second cistron) (Fig. 1b). Triplet pe-
riodicity of the riboseq signal is consistent with trans-
lation of these reading frames. The ASDURF gene is
translated by one order of magnitude more efficiently
than the ASNSD1, which, likely, indicates that transla-
tion of the first cistron strongly repress translation of
the second cistron (Fig. 1b). Interestingly, however, in
the experiments with reporter constructs mutation of
the ASDURF start codon derepresses translation of the
luciferase fused to the second cistron only ~2 fold [39],
which is less than expected. This suggests that when
the AUG of ASDURF is mutated, the ribosomes begin
to initiate translation at the AUG codons within the
ASDURF reading frame that are normally not recog-
nized, when the ASDURF start site is active.
Phylogenetic analysis reveals that the sequence
of ASDURF corresponding to the 1st exon is con-
served in mammals, while the 2nd and 3rd ex-
ons show high rate of synonymous codon substitu-
tions also across the vertebrate genomes (Fig. 1c).
ANDREEV, SHATSKY34
BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025
Fig. 1. a) Aggregated Riboseq and RNAseq data for the ASNSD1 gene from the GWIPS-Viz browser [29]. Nucleotide evolu-
tionary conservation is shown as 100 vertebrates Basewise Conservation by PhyloP (phyloP100way) track [30]. b)Aggregated
Riboseq data from the Ribocrypt transcriptome browser (ribocrypt.org, in preparation), ribosome footprints are color-coded
according to the translated reading frames shown below in the diagram. c) Multiple sequence alignment performed with
CodAlignView (“CodAlignView: A tool for Exploring Signatures of Protein-Coding Evolution in an Alignment”, Jungreis, I.,
Lin, M., and Kellis, M., in preparation), the alignment set used was hg30_100. d) Structures and alignment of human, chick-
en, and zebrafish ASDURF. Structures were generated using AlphaFold 3 [31]. Sequence alignment was performed with
ClustalW [32]. e) Schematic representation of the ASDURF-ASNSD1 bicistronic mRNA. AUG codons upstream of the second
cistron are marked with colored arrows. The proposed functions of proteins encoded in cistrons are presented below.
BICISTRONIC mRNAs IN MAMMALS 35
BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025
Analysis of the chicken and zebrafish ASNSD1 genes
shows that these organisms use alternative first ex-
ons, which contain an in-frame AUG codon. Due to
this, the N-terminal portion of ASDUSF is variable.
However, according to the AlphaFold 3 modelling,
these proteins may have a similar structural orga-
nization consistent with the b-prefoldin structure
(Fig. 1d).
How can translation of the ASDURF-ASNSD1
bicistronic mRNA be regulated? To investigate ef-
ficiency of AUG codons, we explored the data from
the quantitative analysis of mammalian translation,
where the strength of AUG contexts from −6 to +5
positions was experimentally measured and quanti-
fied [40]. The AUG contexts efficiency ranges from 12
(least efficient) to 150 (most efficient). Interestingly,
all the AUGs upstream of the ASNSD1 CDS including
the AUG of the ASDURF have suboptimal or weak
contexts (Fig. 1e). Based on this information, we hy-
pothesize that the ASNSD1 translation may be regu-
lated under conditions of altered stringency of start
codon selection, or when the translation elongation
rate is regulated. Increasing initiation at suboptimal
AUG codons or slowing down the elongating ribosome
that translates ASDURF is expected to significantly
reduce the downstream translation of ASNSD1.
SLC35A4URF-SLC35A4
The SLC35A4 gene locus encodes a protein
that was initially predicted to have a pyrimidine
nucleotide-sugar transmembrane transport activi-
ty. In addition, there is a short protein-coding gene
ENSG00000293600 located upstream of the SLC35A4
CDS. In parallel with other cases described here, we
suggest calling this upstream open reading frame
SLC35A4URF. SLC35A4URF could be translated either
as a separate ORF, due to the use of alternative polya-
denylation site between SLC35A4 and SLC35A4URF, or
as a bicistronic mRNA. Based on the cap analysis of
gene expression (CAGE) data from the FANTOM5 proj-
ect [22] (visualized in the Zenbu browser [23]), it ap-
pears that SLC35A4 could only be translated from the
bicistronic transcripts (Fig. 2a, Fig. S1B in the Online
Resource 1).
In 2015, we predicted that the upstream open
reading frame in the SLC35A4 gene could encode a
functional protein [41]. Functional role of SLC35A4URF
or SLC35A4-MP has been the subject of two recent
studies, and it has been shown that it plays a role
in mitochondria [42, 43]. Yang et al. [43] demonstrated
that the SLC35A4URF protein is localized in mitochon-
dria. Using co-immunoprecipitation mass spectrometry
(co-IP/MS) and Western blotting, they demonstrated
that the SLC35A4URF interacts with the mitochondrial
outer membrane proteins, such as VDAC1 andVDAC3.
Additionally, co-immunostaining experiments re-
vealed that the SLC35A4URF was colocalized with
the outer membrane marker TOMM20. Rocha et al.
[42] developed rabbit polyclonal antibodies against
SLC35A4URF and demonstrated that the endogenous
protein is located in mitochondria. However, based on
the proteinase K assays, SLC35A4ORF appears to be
a mitochondrial inner membrane protein rather than
an outer membrane protein. These results are con-
sistent with the presence of a single-pass transmem-
brane domain between the amino acids 62 and 84
of SLC35A4URF [42].
Functional role of SLC35A4URF has been shown to
be linked to mitochondrial respiration. Using genome
editing, Rocha et al. generated the KO HEK293T cell
lines and demonstrated that the loss of this protein
resulted in significant decrease in basal proton leak
and maximal respiration. Conversely, overexpression
of the SLC35A4URF gene leads to the increase in maxi-
mal capacity rates. Interestingly, 3-fold overexpression
of SLC35A4URF in the MCF7 cells impairs cell growth,
which was accompanied by the loss of mitochondrial
membrane potential and upregulation of transcrip-
tion of the genes enriched in the mitochondria-re-
lated pathways such as “response to hypoxia” and
“negative regulation of mitochondrial membrane po-
tential”. This suggests that the amount of SLC35A4URF
protein must be tightly regulated in order to
promote optimal cellular growth.
Function of the second cistron product, SLC35A4,
is related to regulation of glycosylation in the Golgi
apparatus. SLC35A4 and its paralog SLC35A1 could
be involved in the transport of CDP-ribitol from the
cytoplasm to Golgi [44]. CDP-ribitol, which is synthe-
tized by the ISPD enzyme in the cytoplasm, is used
for assembly of a complex glycan on the α-dystro-
glycan glycoprotein by the FKTN and FKRP enzymes
in the Golgi apparatus [45]. Modification with the
ribitol moieties is required for proper interaction
of α-dystroglycan with extracellular matrix proteins
such as laminin. Localization of the overexpressed
SLC35A4 in the Golgi was experimentally confirmed
by Rocha et al. [42].
The aggregated riboseq data for the human
SLC35A4URF-SLC35A4 mRNA reveals efficient transla-
tion of the first cistron and almost undetectable trans-
lation of the second cistron (Fig.2,a and b). The first
312-nt-long ORF is followed by the long spacer region
of 407 nucleotides that contains several AUG codons
(11 in total preceding the second cistron). Phyloge-
netic analysis reveals that this spacer region is the
least conserved in the vertebrate genomes (Fig. 2d).
Interestingly, the riboseq data from zebrafish, where
the spacer region is much shorter, reveals translation
of both cistrons (Fig. 2c).
ANDREEV, SHATSKY36
BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025
Fig. 2. a)Aggregated Riboseq and RNA-seq data for the SLC35A4 gene from the GWIPS-Viz browser [29]. Nucleotide evolution-
ary conservation is displayed as 100 vertebrates Basewise Conservation by PhyloP (phyloP100way) track [30]. b)Aggregated
Riboseq data from the Ribocrypt transcriptome browser (ribocrypt.org, in preparation). Ribosome footprints are color-coded
according to the translated reading frames shown below in the diagram. c) Aggregated Riboseq and RNAseq data for the
zebrafish SLC35A4 gene from the GWIPS-Viz browser [29]. d) Multiple sequence alignment performed with CodAlignView
(“CodAlignView: A tool for Exploring Signatures of Protein-Coding Evolution in an Alignment”, Jungreis, I., Lin, M., and
Kellis, M., in preparation), the alignment set used is hg30_100. e) Schematic representation of the SLC35A4URF-SLC35A4
bicistronic mRNA. AUG codons located upstream of the second cistron are marked with colored arrows. Possible functions
for the proteins encoded in both cistrons are shown below.
BICISTRONIC mRNAs IN MAMMALS 37
BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025
Regulation of translation of the SLC35A4URF-
SLC35A4 bicistronic mRNA during the integrated
stress response has been discovered in two indepen-
dent studies [41, 46]. During the integrated stress re-
sponse (ISR), translation of the SLC35A4 increases sig-
nificantly, which is accompanied by downregulation
of the first cistron. Andreev et al. [41] performed a
reporter analysis with the reporter mRNA, where the
second cistron has been replaced with firefly lucifer-
ase. Under normal conditions, this capped bicistron-
ic mRNA produced very low but detectable levels of
reporter activity, which was approximately two or-
ders of magnitude lower than that for the reporter
mRNA with the short 5′-leader. After treatment with
arsenite, which activates eIF2 phosphorylation, trans-
lation of the second cistron increased, while trans-
lation of the control mRNA decreased approximate-
ly 10-fold. Therefore, the sequence upstream of the
SLC35A4 second cistron appears to be sufficient to
mediate stress resistant translation. Sequence analysis
reveals that all 11 AUG codons upstream of the sec-
ond cistron contain non-optimal nucleotide contexts
(Fig. 2e), which raises the possibility that translation
of the second cistron could be mediated by leaky
scanning.
Taken together, translation control of SLC35A4URF-
SLC335A4 could connect mitochondrial respiration
with extracellular matrix. During the integrated stress
response, translation of the first cistron is decreased,
which apparently should lead to the decrease in mito-
chondrial respiration. At the same time, the stress-in-
duced activation of translation of SLC35A4 could sup-
port maturation of α-dystroglycan. It is worth noting
that the SLC35A1 translation, which is also involved
in the CDP-ribitol transport, is significantly downreg-
ulated during the arsenite-induced stress [41]. Inter-
estingly, when SLC35A4 is overexpressed in the cells
lacking SLC35A1, glycosylation of α-dystroglycan is
rescued, but molecular weight of the glycosylated
form of α-dystroglycan is significantly decreased [44].
This indicates that SLC35A4 could alter glycosylation
pattern of its substrate. This allows us to speculate
that under stress conditions, the α-dystroglycan glyco-
sylation pattern could be altered due to the changes
in the ratio of SLC35A4 to SLC35A1.
MIURF-MIEF1
The MIEF1 gene locus was suggested to encode
a bicistronic mRNA based on phylogenetic analysis
of its upstream uORF [41]. The product of the sec-
ond cistron, MIEF1 protein, plays a role in regulating
mitochondrial fusion and fission. Additionally, there
is a micropeptide called MIURF encoded in the first
cistron (ENSG00000285025) that operates in mitochon-
dria and, likely, also regulates MIEF1. According to the
cap analysis of gene expression (CAGE) data from the
FANTOM5 project [22] (visualized in the Zenbu brows-
er [23]), both MIURF and MIEF1 share a common pro-
moter (Fig. S1C in the Online Resource 1).
MIEF1 (a.k.a. MID51) and MIEF2 (a.k.a. MID49)
are two closely related proteins that were discovered
in 2011. These proteins are involved in regulation of
mitochondrial dynamics, as they can change morphol-
ogy of mitochondria (fission and fusion), when they
are overexpressed or knocked out [47]. MIEFs are out-
er mitochondrial membrane proteins, which contain a
single-pass transmembrane domain at the N-terminus
with bulk of the protein facing the cytosol. MIEFs act
as a receptor hub that recruits both pro-fusion pro-
teins MFN1 and MFN2, and pro-fission protein DRP1
[48]. It was shown that MIEFs can form oligomers
and self-associate, which could promote fusion of the
adjacent mitochondria [48].
MIURF, a protein encoded in the first cistron, ap-
pears to have two mutually exclusive functions: regu-
lation of mitochondrial translation, which takes place
in the mitochondrial matrix, and regulation of MIEF1
activity on the cytoplasmic side of the mitochondrial
outer membrane. Initially, MIURF was discovered as
a component of the mitochondrial ribosome assem-
bly intermediate for the large ribosomal subunit, LSU.
Mitoribosomal assembly intermediates were isolated
from the human cell line derived from HEK293S cells
and analyzed using cryo-EM. The authors observed
a density adjacent to uL14m and used the density-
based fold-recognition pipeline and mass-spectrome-
try analysis to identify three proteins: MALSU, mt-ACP,
and MIURF (known as L0R8F8 at the time of publi-
cation). This module was proposed to prevent pre-
mature association of the ribosomal subunits [49].
Rathore etal. [50] supported the role of MIURF in the
regulation of mitochondrial translation. Using various
approaches, the authors demonstrated that MIURF
interacts with the mitochondrial ribosome and that
MIURF positively regulates mitochondrial translation.
In both studies mitochondrial ribosomes were shown
to interact with the endogenous MIURF, which strong-
ly supports its functional role in translation. Whether
MIURF serves exclusively as a mitoribosome assembly
factor, or is it also a component of the mature trans-
lation machinery, remains to be determined.
Another activity of MIURF was discovered in
2020. Chen et al. [51] performed genome-wide CRISPR
screening to identify non-canonical CDSs that influ-
ence cellular growth; and this approach allowed them
to identify MIURF as one of the top hits. They further
showed that the overexpressed tagged MIURF physi-
cally interacts with the second cistron product, MIEF1.
Importantly, the tagged MIEF1 pulls down MIURF.
Interestingly, overexpression of MIURF induces
ANDREEV, SHATSKY38
BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025
Fig. 3. a)Aggregated Riboseq and RNAseq data for the MIEF1 gene from the GWIPS-Viz browser [29]. Nucleotide evolutionary
conservation is shown as the 100 vertebrates Basewise Conservation by PhyloP (phyloP100way) track [30]. b) Aggregated
Riboseq data from the Ribocrypt transcriptome browser (ribocrypt.org, in preparation). Ribosome footprints are color-cod-
ed according to the translated reading frames shown below the diagram. c) Multiple sequence alignment performed with
CodAlignView (“CodAlignView: A tool for Exploring Signatures of Protein-Coding Evolution in an Alignment”, Jungreis, I.,
Lin, M., and Kellis, M., in preparation), the alignment set used is hg30_100. d)Schematic representation of the MIURF-MIEF1
bicistronic mRNA. AUG codons located upstream of the second cistron are marked with colored arrows. Possible functions
for the proteins encoded in both cistrons are shown below.
BICISTRONIC mRNAs IN MAMMALS 39
BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025
afragmented mitochondrial phenotype (increased fis-
sion), while knockout of MIURF results in increased
fusion, which can be rescued by exogenous expression
of MIURF. These findings suggest that MIURF could
influence activity of MIEF1 by modulating its interac-
tion with the fusion/fission machinery on the cytosolic
side of the mitochondrial outer membrane.
To understand how MUIRF-MIEF1 translation
can be regulated, we analyzed riboseq data (Fig. 3, a
and b) and performed phylogenetic analysis (Fig. 3c).
The aggregated Riboseq data supports translation of
both cistrons from the bicistronic mRNA. In humans,
bisictronic mRNA contains five AUG codons upstream
of the MIEF1 CDS. The 3rd AUG in a suboptimal nucle-
otide context initiates translation of the MIURF coding
sequence. Analysis of the vertebrate genomes reveals
that the MIURF reading frame is conserved among
vertebrates, and the spacer region between MIURF
and MIEF1 is less conserved. All five upstream AUG
codons are in suboptimal contexts, raising the possi-
bility that translation of the second cistron could be
regulated by leaky scanning (Fig. 3d).
Interestingly, translation of MIURF and MIEF1
has been shown to be differentially regulated under
the ISR induced by arsenite treatment [41]. The ISR
conditions have significantly decreased the ratio of
MIURF to MIEF1 translation. This finding suggests
that the ISR-mediated translation control of MIURF
and MIEF1 could influence various mitochondrial ac-
tivities. Under normal conditions, MIURF is produced
at higher levels than MIEF1. Proteomic analysis per-
formed in [52] revealed that the ratio of MIURF/MIEF
proteins under normal growth conditions in the HEK
293, HeLa, and human colon tissue cells is 2.71, 5.73,
and 2.62, respectively. As MIURF is synthesized in the
cytosol, it has an opportunity to bind immediately to
MIEF1, which is localized on the outer mitochondri-
al membrane (OMM). Excess of MIURF is expected to
be transported to the mitochondrial matrix for mito-
chondrial ribosome biogenesis. Under stress condi-
tions, the ratio of MIURF to MIEF1 decreases. If all
MIURFs become trapped in the complexes with MIEF1,
mitochondrial ribosomal biogenesis would be blocked.
However, this hypothetical mechanism requires exper-
imental verification.
DISCUSSION
Here, we present analysis of the recent data on
translational regulation and function of three bicis-
tronic messenger RNAs that are evolutionarily con-
served in vertebrates. Interestingly, all three cases
show that the two cistrons play distinct functional
roles. This raises the question of how these bisic-
tronic genes evolved. Notably, all three genes have
paralogous second cistron genes (ASNS, SLC35A1, A2,
A3, A5, and MIEF2) that do not contain correspond-
ing uORFs. This suggests that ASNSD1, SLC35A4, and
MIEF1 may have originated from the gene duplication
events followed by fusion to the first cistron, which
initially operated as an individual gene. These fusions
created the translationally controlled regulatory cir-
cuits that benefitted organism fitness and were fixed
in evolution. Interestingly, the intercistronic sequenc-
es that are expected to regulate translation, are less
conserved than the cistrons themselves. This implies
that these sequences have evolved differently to ad-
just translational control. This can be illustrated by
the SLC35A4 gene in humans, which has a much lon-
ger spacer and contains more AUG codons compared
to the zebrafish gene (5 vs. 1 AUG).
Unusual bicistronic organization suggests that
two cistrons are under translation control. Changes in
activity of the translation machinery can quickly alter
the ratio of polypeptides encoded in the upstream and
downstream open reading frames. Indeed, two out of
three mRNAs described here showed these chang-
es during the integrated stress response, when the
eIF2-tRNAi-GTP (TC) concentration becomes limited.
It is highly likely that translation regulation of these
mRNAs is not limited to the integrated stress response.
We noticed that in all three cases, AUG initiation co-
dons located upstream of the second cistron have
non-optimal nucleotide contexts, raising the possibility
of regulation through alterations in the start codon se-
lection stringency. Key players in this process are eIF1
and eIF5 [53-59]. Increased levels of eIF1 decrease
initiation at suboptimal codons, while eIF5 has the
opposite effect. We assume that these factors would
significantly influence translation of the bicistronic
mRNAs described in this study. Another potential reg-
ulator of bicistronic mRNA translation is eIF4G2, also
known as DAP5. Several recent studies have shown
that eIF4G2 regulates translation of the uORF-contain-
ing mRNAs by either promoting reinitiation or leaky
scanning[60-65]. In particular, eIF4G2 has been shown
to regulate translation of the dual-coding POLG mRNA,
where initiation at the highly efficient CUG codon al-
lows translation of the POLGARF protein [66]. It would
be extremely interesting to identify the factors that
could change the ratio of translation of the two cis-
trons for ASNSD1, SLC35A4, and MIEF1.
The number of annotated bicistronic mRNAs con-
tinue to increase. For instance, 25 uORFs were recent-
ly annotated as new protein coding genes [67]. Func-
tional investigation of these uORF-encoded proteins
would help us to better understand genome evolution
and the coupling of various cellular processes medi-
ated by the translation control of bicistronic mRNAs.
This is exemplified by the cases of ASNSD1, SLC35A4,
and MIEF1, which are discussed in this paper.
ANDREEV, SHATSKY40
BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025
Supplementary information. The online version
contains supplementary material available at https://
doi.org/10.1134/S0006297924603630.
Contributions. Dmitry E. Andreev conceived the
study, analyzed the data, and wrote the manuscript;
Dmitry E. Andreev and Ivan N. Shatsky edited the
manuscript.
Funding. The work was supported by the Russian
Science Foundation (project no.20-14-00121).
Ethics approval and consent to participate.
This work does not contain any studies involving hu-
man and animal subjects.
Conflict of interest. The authors of this work de-
clare that they have no conflicts of interest.
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