key: cord-0300756-18ge5h4n authors: Minati, Luca; Firrito, Claudia; Piano, Alessia Del; Peretti, Alberto; Sidoli, Simone; Peroni, Daniele; Belli, Romina; Gandolfi, Francesco; Romanel, Alessandro; Bernabò, Paola; Zasso, Jacopo; Quattrone, Alessandro; Guella, Graziano; Lauria, Fabio; Viero, Gabriella; Clamer, Massimiliano title: One-shot analysis of translated mammalian lncRNAs with AHARIBO date: 2020-04-20 journal: bioRxiv DOI: 10.1101/2020.04.20.050062 sha: 318048ba6774c8a5b3dca9c662a17209f420b139 doc_id: 300756 cord_uid: 18ge5h4n A vast portion of the mammalian genome is transcribed as long non-coding RNAs (lncRNAs) acting in the cytoplasm with largely unknown functions. Surprisingly, lncRNAs have been shown to interact with ribosomes, encode uncharacterized proteins, or act as ribosome sponges. These functions still remain mostly undetected and understudied owing to the lack of efficient tools for genome-wide simultaneous identification of ribosome-associated lncRNAs and peptide-producing lncRNAs. Here we present AHARIBO, a method for the detection of lncRNAs either untranslated, but associated with ribosomes, or encoding small peptides. Using AHARIBO in mouse embryonic stem cells during neuronal differentiation, we isolated ribosome-protected RNA fragments, translated RNAs and corresponding de novo synthesized polypeptides. Besides identifying mRNAs under active translation and associated ribosomes, we found and distinguished lncRNAs acting as ribosome sponges or encoding micropeptides, laying the ground for a better functional understanding of hundreds lncRNAs. , R E S U L T S N a s c e n t p e p t i d e l a b e l l i n g a n d s e p a r a t i o n o f t h e r i b o s o m e c o m p l e x w i t h A H A R I B O -r C T o s i m u l t a n e o u s l y p u r i f y r i b o s o m e s u n d e r a c t i v e t r a n s l a t i o n , a s s o c i a t e d R N A s a n d c o r r e s p o n d i n g g r o w i n g p e p t i d e c h a i n s , w e o p t i m i z e d a p r o t o c o l i n H e L a c e l l s ( F i g 1 A ) . T o m i n i m i z e t h e a m o u n t o f A H A -t a g g e d p r o t e i n s a l r e a d y r e l e a s e d f r o m r i b o s o m e s a n d a c h i e v e o p t i m a l o n -r i b o s o m e p o l y p e p t i d e s t a b i l i z a t i o n , w e t e s t e d d i f f e r e n t t i m e s o f A H A i n c u b a t i o n a n d c o m p a r e d t h e e f f e c t o f t w o s m a l l m o l e c u l e s ( n a m e l y c y c l o h e x i m i d e ( C H X ) a n d s B l o c k , a n a n i s o m y c i n -b a s e d r e a g e n t ) , w h i c h a r e k n o w n t o i n h i b i t t h e a c t i v i t y e u k a r y o t i c r i b o s o m e , w h i l e k e e p i n g p o l y p e p t i d e s b o u n d t o t h e t r a n s l a t i n g r i b o s o m e s ( G a r r e a u d e L o u b r e s s e e t a l . , 2 0 1 4 ; G r o l l m a n , 1 9 6 7 ; S e e d h o m e t a A p r o t e i n c o n t e n t o f e a c h i n d i v i d u a l f r a c t i o n w a s v i s u a l i z e d a f t e r S D S -P A G E . S t a i n i n g o f t h e m e m b r a n e w a s p e r f o r m e d b y b i o t i n c y c l o a d d i t i o n f o l l o w e d b y s t r e p t a v i d i n -H R P . R P L 2 6 p r o t e i n w a s u s e d a s r i b o s o m e m a r k e r i n t h e d i f f e r e n t f r a c t i o n s . C ) B o x p l o t s h o w i n g t h e A H A s i g n a l e n r i c h m e n t i n t h e p o l y s o m a l r e g i o n o f t h e p r o f i l e s o b t a i n e d f r o m c e l l s t r e a t e d w i t h e i t h e r C H X o r s B l o c k o r n o d r u g s ( N T ) . R e s u l t s a r e s h o w n a s a m e d i a n ( ± S E ) o f 3 i n d e p e n d e n t r e p l i c a t e s . D ) V o l c a n o p l o t s s h o w i n g t h e -L o g ( P -v a l u e ) v e r s u s t h e r e l a t i v e a b u n d a n c e o f A H A R I B O -r C -i s o l a t e d p r o t e i n s . 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