The nucleoplasmic phase of pre-40S formation prior to nuclear export

Abstract

Biogenesis of the small ribosomal subunit in eukaryotes starts in the nucleolus with the formation of a 90S precursor and ends in the cytoplasm. Here, we elucidate the enigmatic structural transitions of assembly intermediates from human and yeast cells during the nucleoplasmic maturation phase. After dissociation of all 90S factors, the 40S body adopts a close-to-mature conformation, whereas the 3' major domain, later forming the 40S head, remains entirely immature. A first coordination is facilitated by the assembly factors TSR1 and BUD23–TRMT112, followed by re-positioning of RRP12 that is already recruited early to the 90S for further head rearrangements. Eventually, the uS2 cluster, CK1 (Hrr25 in yeast) and the export factor SLX9 associate with the pre-40S to provide export competence. These exemplary findings reveal the evolutionary conserved mechanism of how yeast and humans assemble the 40S ribosomal subunit, but reveal also a few minor differences.

Impact of the yeast S0/uS2-cluster ribosomal protein rpS21/eS21 on rRNA folding and the architecture of small ribosomal subunit precursors

“…Corresponding densities were only scarcely visible in the Enp1TAP_A map. This newly mapped N-terminal portion of yeast Tsr1 partly overlaps with the recently observed position of the human Tsr1 N-terminus, but the described extension towards the immature helix h28 was not detected in the Enp1TAP-S21_A map [ 22 ].…”

“…We consider that this specific, apparently small sub-population of S0-cluster depleted Slx9 associated SSU particles additionally contains Rrp12 and Bud23. The human counterparts of all three proteins interact with the SSU head [ 22 ], and their presence could thereby be obscured in the Slx9TAP-S21_A particle population due to the ill-defined head domain orientation or architecture.…”

“…In yeast, this frameshift is reversed at the third position by the formation of a new base pair with nucleotide U9 which is unpaired in the canonical CPK. Reinspection of recent cryo-EM derived maps and models of human late nuclear to early cytoplasmic SSU precursors indicates that the human CPK also exists in an immature state with the very same frameshift [ 22 ]. In this case, the frameshift is reversed by an unpaired nucleotide at position 3, which is different from the new base pair formed at this site in yeast with nucleotide U9.…”

“…Instead, a new group of factors associates with the SSU precursor. Combined biochemical and structural data support that this group includes the endonuclease Nob1, Tsr1, Rio2, Bud23, Slx9, and Rrp12 together with Pno1, Dim1, and Enp1 which are retained from previous states [ 18 , 22 ]. Different from their human counterparts [ 23 – 25 ], yeast Nob1, Slx9, and Rrp12 were barely detected at any structural snapshot [ 26 – 30 ].…”

“…In addition, a cluster of the three r-proteins rpS0/uS2, rpS2/uS5, and rps21/eS21 (subsequently termed S0-cluster) is now detected near the newly formed CPK and, in human particles, near Nob1. S0-cluster formation might precede Nob1 integration, as suggested by a structural state unexpectedly observed in human particles associated with a dominant negative mutant form of the late acting factor human RIOK1 (named Rio1 in yeast) [ 22 ]. The wild-type form of RIOK1 is only detected in late cytoplasmic SSU precursors after the release of most of the previous factors.…”