A conserved structural element in the RNA helicase UPF1 regulates its catalytic activity in an isoform-specific manner

Abstract

The RNA helicase UPF1 is a key component of the nonsense mediated mRNA decay (NMD) pathway. Previous X-ray crystal structures of UPF1 elucidated the molecular mechanisms of its catalytic activity and regulation. In this study, we examine features of the UPF1 core and identify a structural element that adopts different conformations in the various nucleotide- and RNA-bound states of UPF1. We demonstrate, using biochemical and single molecule assays, that this structural element modulates UPF1 catalytic activity and thereby refer to it as the regulatory loop. Interestingly, there are two alternatively spliced isoforms of UPF1 in mammals which differ only in the lengths of their regulatory loops. The loop in isoform 1 (UPF11) is 11 residues longer than that of isoform 2. We find that this small insertion in UPF11 leads to a two-fold increase in its translocation and ATPase activities. To determine the mechanistic basis of this differential catalytic activity, we have determined the X-ray crystal structure of the helicase core of UPF11 in its apo-state. Our results point toward a novel mechanism of regulation of RNA helicases, wherein alternative splicing leads to subtle structural rearrangements within the protein that are critical to modulate enzyme movements and catalytic activity.

UPF1-Mediated RNA Decay—Danse Macabre in a Cloud

“…Central to both models is UPF1, a 123 kDA ATP-dependent 5′–3′ RNA/DNA-helicase and prototype member of the SF1 superfamily (also called UPF-like helicases [ 46 ]). UPF1 consists of an unstructured N-terminal region, a cysteine–histidine-rich (CH) zinc-knuckle domain, a helicase region and a carboxy-terminal serine and glutamine rich (SQ) domain.…”

“…A pocket-like structure between RecA1 and RecA2 binds ATP [ 49 , 50 , 51 , 52 ]. The UPF1 helicase domain contains two additional regulatory subdomains 1B and 1C that effect UPF1′s high processivity and exert regulatory functions [ 46 , 47 , 52 , 53 ]. Alternative splicing results in two UPF1 isoforms (UPF1 1 and UPF1 2 ) that differ from each other exclusively by an 11-amino-acid insertion within subdomain B which confers enhanced RNA binding and catalytic activity to the UPF1 1 variant [ 46 ].…”

“…The UPF1 helicase domain contains two additional regulatory subdomains 1B and 1C that effect UPF1′s high processivity and exert regulatory functions [ 46 , 47 , 52 , 53 ]. Alternative splicing results in two UPF1 isoforms (UPF1 1 and UPF1 2 ) that differ from each other exclusively by an 11-amino-acid insertion within subdomain B which confers enhanced RNA binding and catalytic activity to the UPF1 1 variant [ 46 ]. The CH domain is an important modulator of UPF1 function and a hotspot of interaction with other proteins ([ 54 ] reviewed in [ 7 ]) ( Figure 1 ).…”

“…While UPF1 can be found on all 3′UTRs, its ATPase activity has been reported to promote UPF1 release preferentially from non-target mRNAs [ 125 ]. ATP binding reduces UPF1′s affinity for RNA and activates its helicase activity [ 46 , 52 , 128 ]. UPF1 mutants unable to bind or hydrolyze ATP display enhanced RNA binding and are locked on target and non-target mRNAs while hyperactive ATPase mutants lose their RNA binding competence.…”

A regulated NMD mouse model supports NMD inhibition as a viable therapeutic option to treat genetic diseases

“…The dnUPF1 transgene is a cDNA and, therefore, is not subject to splicing. However, endogenous UPF1 has been reported to be expressed as multiple isoforms in various tissues (Gowravaram et al, 2018). Thus, although some of these bands could potentially represent a nonspecific protein product, they most likely represent unique, tissue-specific UPF1 isoforms that may or may not also have differential post-translational modifications (Popp and Maquat, 2015).…”