UCHL1 [10]. More recently, Tran et al. [11] reported a
NAT-lncRNA, named AS-RBM15 (antisense RNA of
RNA-binding protein 15 involved in megakaryocyte differentiation), which enhances the translation of the RBM15 in a
cap-dependent manner during megakaryocyte differentiation and
is also involved in a chromosome translocation in acute megakaryocyte leukemia.
The role of lncRNAs in translation has also been elucidated in
the plant kingdom. A cis-NAT lncRNA (i.e., a NAT that acts on the
transcript originated from the opposite strand) enhances the translation of its cognate sense mRNA, the PHOSPHATE 1;2, contributing to maintain phosphate homeostasis in rice during phosphate
deficiency [12]. Another three cis-NAT lncRNAs associated with
ribosomes (ribo-cis-NATs) have been found in Arabidopsis, which
also showed a positive correlation with translation of their sense
transcripts in plants subjected to phosphate limitation [13]. The
sense mRNAs displaying NAT regulation encode ATP-BINDING
CASSETTE SUBFAMILY G transporters (ABCG2 and ABCG20)
and a POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 7
(PRK7) family member. More recently, Deforges et al. [14] used
RNA-seq and translatome (i.e., the population of transcripts associated with actively translating ribosomes) data obtained in Arabidopsis to identify ribo-cis-NAT RNAs whose levels are correlated
(positively or negatively) with their sense transcripts across several
experimental conditions. Fourteen cis-NAT RNAs were identified,
and some were validated using transgenic plants and protoplasts,
showing that translatome datasets can be used to systematically
identify cis-NAT RNAs that regulate translation.
Association of lncRNAs with the translational machinery has
been revealed by different experimental approaches that include
differential ultracentrifugation through sucrose density gradients,
ribosome footprinting (Ribo-seq), translating ribosome affinity
purification (TRAP), or a combination of these approaches [29–
35]. In this chapter, we focus on the protocol of TRAP combined
with direct RNA sequencing (RNA-seq) and ulterior bioinformatics analysis used to detect lncRNAs associated with actively translating ribosomes. The TRAP methodology is based on the
expression of a cytosolic ribosomal protein fused to an epitope
(FLAG, c-myc, or HA) or a fluorescent protein (e.g., GFP, RFP,
YFP) in the tissues or specific cells of interest [36, 37]. The fusion
protein is incorporated into ribosomes and polysomes, allowing the
isolation of these ribonucleoprotein (RNPs) complexes by immunopurification using specific antibodies coupled to magnetic or
agarose beads (Fig. 3a). The TRAP approach has the advantage of
excluding RNPs other than ribosomes and polysomes, which due
to similar sedimentation coefficient are usually co-purified when
differential ultracentrifugation is used. TRAP RNA is purified using
standard RNA purification procedures and can subsequently be
454
Soledad Traubenik et al.
NAT-lncRNA, named AS-RBM15 (antisense RNA of
RNA-binding protein 15 involved in megakaryocyte differentiation), which enhances the translation of the RBM15 in a
cap-dependent manner during megakaryocyte differentiation and
is also involved in a chromosome translocation in acute megakaryocyte leukemia.
The role of lncRNAs in translation has also been elucidated in
the plant kingdom. A cis-NAT lncRNA (i.e., a NAT that acts on the
transcript originated from the opposite strand) enhances the translation of its cognate sense mRNA, the PHOSPHATE 1;2, contributing to maintain phosphate homeostasis in rice during phosphate
deficiency [12]. Another three cis-NAT lncRNAs associated with
ribosomes (ribo-cis-NATs) have been found in Arabidopsis, which
also showed a positive correlation with translation of their sense
transcripts in plants subjected to phosphate limitation [13]. The
sense mRNAs displaying NAT regulation encode ATP-BINDING
CASSETTE SUBFAMILY G transporters (ABCG2 and ABCG20)
and a POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 7
(PRK7) family member. More recently, Deforges et al. [14] used
RNA-seq and translatome (i.e., the population of transcripts associated with actively translating ribosomes) data obtained in Arabidopsis to identify ribo-cis-NAT RNAs whose levels are correlated
(positively or negatively) with their sense transcripts across several
experimental conditions. Fourteen cis-NAT RNAs were identified,
and some were validated using transgenic plants and protoplasts,
showing that translatome datasets can be used to systematically
identify cis-NAT RNAs that regulate translation.
Association of lncRNAs with the translational machinery has
been revealed by different experimental approaches that include
differential ultracentrifugation through sucrose density gradients,
ribosome footprinting (Ribo-seq), translating ribosome affinity
purification (TRAP), or a combination of these approaches [29–
35]. In this chapter, we focus on the protocol of TRAP combined
with direct RNA sequencing (RNA-seq) and ulterior bioinformatics analysis used to detect lncRNAs associated with actively translating ribosomes. The TRAP methodology is based on the
expression of a cytosolic ribosomal protein fused to an epitope
(FLAG, c-myc, or HA) or a fluorescent protein (e.g., GFP, RFP,
YFP) in the tissues or specific cells of interest [36, 37]. The fusion
protein is incorporated into ribosomes and polysomes, allowing the
isolation of these ribonucleoprotein (RNPs) complexes by immunopurification using specific antibodies coupled to magnetic or
agarose beads (Fig. 3a). The TRAP approach has the advantage of
excluding RNPs other than ribosomes and polysomes, which due
to similar sedimentation coefficient are usually co-purified when
differential ultracentrifugation is used. TRAP RNA is purified using
standard RNA purification procedures and can subsequently be
454
Soledad Traubenik et al.