Science - 06.03.2020

(Barry) #1
INSIGHTS | PERSPECTIVES

sciencemag.org SCIENCE

GRAPHIC: KELLIE HOLOSKI/

SCIENCE

By Lian-Huan Wei and Junjie U. Guo

H

igh-throughput RNA sequencing
studies have revealed pervasive
transcription of the human ge-
nome, which generates a variety of
long noncoding RNAs (lncRNAs)
that have no apparent protein-
coding functions ( 1 ). Subsequent studies
that globally monitor translation have
similarly identified numerous transla-
tion events outside of canoni-
cal protein-coding sequences
( 2 – 4 ), suggesting pervasive
translation of the transcrip-
tome. However, only a few ex-
amples of functional peptides
encoded by RNA regions previ-
ously thought to be noncoding
have been reported to regulate
distinct biological processes
( 5 – 9 ). On page 1140 of this is-
sue, Chen et al. ( 10 ) provide
evidence for an expanded rep-
ertoire of functional peptides
encoded by lncRNAs and other
“untranslated” RNA regions.
Sequencing of ribosome-pro-
tected messenger RNA (mRNA)
fragments (RPFs) allows re-
searchers to globally identify
RNA regions that are trans-
lated (open reading frames,
or ORFs). In addition to the
canonical open reading frames
(main ORFs) of mRNAs, RPFs
have also been found in the
untranslated regions (UTRs) of
mRNAs and in many lncRNAs
( 2 – 4 ). Although some RPFs may
not represent bona fide transla-
tion ( 11 ), the question remains
whether translation of lncRNAs
and UTRs is functional or rep-
resents an inconsequential
amount of translation that occurs on all cy-
tosolic 5 9 -capped RNAs (see the figure).
Chen et al. used genome-wide loss-of-
function screens to systematically assess
the functionality of noncanonical ORFs
in cell growth. They first filtered RPFs
obtained from several human cell types

and identified more than 5000 previ-
ously unannotated ORFs that showed ro-
bust evidence of active translation. These
newly identified ORFs include variants
of canonical ORFs (e.g., canonical ORFs
with an alternative initiation position),
upstream ORFs (uORFs) within 5 9 UTRs,
and ORFs within transcripts that are an-
notated as lncRNAs. Consistent with previ-
ous studies, these ORFs are shorter (fewer
than 100 amino acids; hence, they are also

called microproteins) and less conserved
than canonical ORFs. They often use near-
cognate translation start codons such as
CUG, whereas canonical ORFs are pre-
dominantly initiated at AUG start codons.
More than 200 newly identified micropro-
teins were validated by orthogonal pro-
teomic and peptidomic analyses in mul-
tiple human cell lines, suggesting that at
least some microproteins accumulate in
cells to detectable concentrations.

Using CRISPR-Cas9, Chen et al. dis-
rupted each of 2353 selected unannotated
ORFs and identified more than 400 ORFs
that promoted cell growth in both human
K562 leukemia cells and induced pluripo-
tent stem cells. In addition to changes in
cell growth, the disruption of many unan-
notated ORFs also caused specific changes
in gene expression. Consistent with trans-
lation being essential, the authors found
that start codons were critical for the func-
tions of a few ORF-containing
lncRNAs. However, among
all the lncRNAs that affected
cell growth when they were
transcriptionally silenced by
CRISPR interference, only a
small subset showed consistent
phenotypes when their embed-
ded ORFs were disrupted, sug-
gesting noncoding functions of
the remaining lncRNA loci.
Peptides encoded by ln-
cRNAs and uORFs within
59 UTRs showed specific sub-
cellular localization and pro-
tein interactions. Notably,
some uORF-encoded peptides
formed complexes with the
proteins encoded by the cor-
responding main ORFs. The
functional importance of such
interactions remains to be
tested, as well as the general-
ity of eukaryotic mRNAs en-
coding multiple subunits of a
protein complex. Also unre-
solved is the extent to which
the growth phenotypes of some
uORFs may be partly due to
their regulation of main ORF
translation and independent of
the uORF-translated peptides
(i.e., translation-dependent,
product-independent), which
is a well-established function of uORFs
( 12 ). Future studies on the genetic inter-
actions between uORFs and main ORFs
within the same mRNAs should provide an
answer. Analogous to the translation regu-
lation function of uORFs, translation may
also regulate the function and stability of
some bona fide lncRNAs by remodeling
RNA structures and/or ribonucleoprotein
composition, a possibility that remains
largely unexplored.

MOLECULAR BIOLOGY

Coding functions of “noncoding” RNAs


Hundreds of RNA regions that encode microproteins are found to regulate cell growth


Department of Neuroscience, Yale University
School of Medicine, New Haven, CT 06520, USA.
Email: [email protected]

AAAAA
AAAAA
No function (translation noise)

AAAAA
AAAAA
Translation-dependent, product-independent function

AAAAA
AAAAA
Product-dependent function

AAAAA
AAAAA

Nucleus Cytoplasm

mRNA lncRNA

uORF Main ORF ORF

Ribosome

1074 6 MARCH 2020 • VOL 367 ISSUE 6482

Possible functions of noncoding RNA translation
Aside from the main open reading frames (ORFs) of messenger RNAs (mRNAs),
translation also occurs in upstream ORFs (uORFs) within 5 9 untranslated
regions (UTRs) as well as ORFs within cytosolic long noncoding RNAs (lncRNAs).
These translation events may either have no function, regulate main ORF
translation, regulate noncoding functions of lncRNAs, produce functional peptides
that interact with main ORF-encoded proteins, or function independently.

Published by AAAS
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