La is the first protein to interact with nascent pre-tRNAs in eukaryotes and remains bound during tRNA processing and modification1,2
. In its best characterized activity, the La domain, comprised of a La motif (LM) and RNA recognition motif (RRM1) in fixed arrangement, protect UUU-3'OH-containing RNAs from 3'-exonucleolytic digestion1–4
Because the LM and RRM1 are required for UUU-3'OH binding5
, it was expected that this binding would involve the β-sheet surface of La RRM16–8
. Surprisingly, a human La (hLa) crystal structure shows that while the LM and RRM1 form a UUU-3'OH binding cleft, most RNA contacts are to the LM, leaving the β-sheet surface of RRM1 unoccupied4
. This documented sequence-specific recognition by a RRM that does not involve its β-sheet surface4
. Solving of four more structures of hLa with different RNAs confirmed the LM mode of recognition with the RRM1 β-sheet surface unoccupied9
. Thus the LM-RRM1 mediates induced fit UUU-3'OH binding9
while leaving the RRM1 β- sheet surface unoccupied and its role in RNA binding unclear3,4,9,10
Some La-related proteins (LARPs) that contain a La domain are telomerase subunits11,12
. LARP7 (a.k.a. PIP7S) is a tumor suppressor that binds 7SK snRNA to regulate P-TEFb13,14
. Ciliate LARPs recognize UUU-3'OH-containing telomerase RNA, and LARP7 recognizes UUU-3'OH of 7SK snRNA, while neither are associated with nascent pre-tRNAs13,15
. Since LARPs have conserved the LM-RRM arrangement they likely use RNA binding modes similar to genuine La. Yet, how the LM and RRM1 β-sheet binding surface work together is not known for any protein.
Some activities of La proteins are more complex than UUU-3'OH binding. Yeast La is required for the maturation of structurally impaired pre-tRNAs16–18
, can promote tRNA folding19
, and acts redundantly with tRNA modification enzymes, consistent with a role in RNA structural integrity20
. Ciliate LARP p65 induces structural rearrangement of telomerase RNA12,21
, and hLa exhibits RNA chaperone activity18,22,23
. While these studies document complex functions related to RNA structure/folding, the mechanisms that distinguish these activities from simple UUU-3'OH binding are unknown. To understand this it will be necessary to know how the LM and RRM interact with RNA dynamically, during simple and complex activities, and in specific pathways of RNA metabolism.
Modified nucleotides found on La-associated pre-tRNAs indicate that La is bound to pre-tRNAs during modification24,25
. La remains associated with pre-tRNA until RNase Z-mediated endonucleolytic cleavage separates the tRNA body from the UUU-3'OH-containing 3' trailer, the latter of which varies for different pre-tRNAs16,17,26–29
. While pre-tRNA processing occurs in minutes, the half life of La is hours24,30
, and although abundant, La can be limiting for tRNA maturation31
. To participate in multiple rounds of tRNA maturation, La must dissociate from the UUU-3'OH-containing trailer products of processing and recycle onto newly synthesized pre-tRNAs. Since cleaved 3' trailers contain UUU-3'OH, the highest affinity sequence-specific ligand known for La, it was unclear how efficient dissociation would occur.
We hypothesized that La might distinguish pre-tRNA from processed products using two binding sites which together provide higher affinity than either alone. After processing, the tRNA and 3' trailer would readily dissociate from their single binding sites. We show that the RRM1 β-sheet surface forms a RNA binding site distinct from the UUU-3’OH binding site that promotes tRNA maturation in vivo. We also show that this RRM1 binding site contributes to the RNA chaperone activity of La.