Science - 06.03.2020

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]

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No function (translation noise)

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Translation-dependent, product-independent function

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Product-dependent function

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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