Here we have analyzed the EM structure of pre-60S particles that contain the Arx1-Alb1 heterodimer. These are relatively late particles of the 60S biogenesis pathway that are eventually exported from the nucleus into the cytoplasm. On the one hand, Arx1-Alb1 is associated with a well-studied nucleoplasmic pre-ribosomal particle that carries the dynein-related AAA+ ATPase Rea1 and the Rix1-complex (Rix1-particle). However, the major pool of Arx1-Alb1 associated pre-60S particles, termed the Arx1-particle, lack Rea1 and Rix1, and represent a biogenesis intermediate that follows the Rix1-particle. The Arx1-particle exhibits characteristic structures such as the foot, knob, and nose, which are prominent features seen both by negative stain and cryo-EM. We show for a few of these structures that they corresponded to additional non-ribosomal densities on the pre-60S particle absent from the mature 60S subunit. Employing immuno-EM, the positions of several ribosomal and non-ribosomal proteins could be mapped. To our knowledge, the Arx1-particle is the first immature 60S subunit precursor for which a cryo-EM structure has been determined.
In our reconstruction, the core of the Arx1-particle is similar to the mature 60S subunit, indicating a late assembly stage. However, several important functional sites of the mature 60S subunit are not yet fully developed on the Arx1-particle. Apparently, the flexible P-stalk is absent from the Arx1-particle. This structure is composed of ribosomal proteins P0 and acidic P1 and P2 on the mature 60S subunit. The P-stalk functions in recruiting translation factors to the GTPase center38
. However, the stalk base seems to be under reconstruction in the Arx1-particle. Mrt4, a P0-paralog that requires the Yvh1 factor for its release from the 60S pre-ribosome39,40
, as well as Yvh1 and P0 are detected in the Arx1-particle by mass-spectrometry. On the other hand, P1 and P2 are known to be late joining r-proteins41
that likely associate with cytoplasmic 60S subunits following release of Arx1-Alb1.
Moreover, the Arx1-purified pre-60S particle differs from the mature subunit in the region of the central protuberance. Together with the observed extra densities, the immature central protuberance gives rise to the characteristic nose
structure of the Arx1-particle, suggesting that final conformational rearrangements of the central protuberance and/or release of ribosome biosynthesis factors from this region may not have yet occurred. The yellow extra density at the central protuberance () may contain part of the structures that form the central protuberance in the mature ribosome, e.g. 5S rRNA, Rpl5, and Rpl11. Possible ribosome biogenesis factors that could contribute to the yellow density are the Mex67-Mtr2 complex or the GTPase Nug1, both of which were shown to interact with 5S rRNA in vitro18,42
. The pre-ribosomal factors responsible for the additional density in the pre-ribosomal structure, other than Tif6 and Arx1-Alb1, have not been unambiguously identified. The localization of the green shape () – albeit smaller – is similar to that found for MPB-tagged Nmd3 on the intersubunit surface of mature 60S in a previous cryo-EM study17
. Interestingly, the purple density stretches far across the pre-ribosomal surface and contacts both Arx1 and the Tif6 density, thus possibly providing a means of direct crosstalk between the distant areas around the stalk base and the exit tunnel. This might enable communication of the state of maturation at the stalk base and the release of Arx1 at the exit tunnel. Likewise, the cyan density might coordinate and communicate the progression of maturation around the stalk base and the tRNA binding sites. Another biogenesis factor Rei1, implicated in release of Arx1-Alb1 from the subunit25,43
, might be represented by the purple density that also contacts Arx1. Other candidate proteins responsible for the extra densities are Ecm1 and several GTPases including Nog1, Nug1, Nog2, and Lsg1 (involved in the release of Nmd344
Notably, all these additional densities on the Arx1-particle were observed at functionally relevant sites of the 60S subunit, blocking access to the ribosomal peptidyl transferase center (PTC), the tRNA binding sites, the stalk base, the intersubunit surface and the exit tunnel. A recent study showed that biogenesis factors present on a late cytoplasmic pre-40S particle block all sites important for translation initiation12
. Similarly, the positioning of biogenesis factors, as well as the immature state of important structures on the Arx1-particle are hindering untimely onset of translation. Furthermore, it was reported that final cytoplasmic pre-40S maturation involves a translation-like cycle45
. It is possible that also pre-60S subunits engage in such a quality control checkpoint to assure functionality and final rRNA maturation and factor release. Together, these observations illustrate the multitude of control mechanisms that keep the nascent subunit from pre-mature engagement in translation during this advanced phase of maturation.
The prominent foot
structure of the Arx1-particle is located above the region where the 3′-end of the 5.8S rRNA and the 5′-end of the 25S rRNA end in a common helix in the mature subunit. Arx1-associated particles mainly contain mature 25S and 5.8S rRNA. However, a small amount of 27SB pre-rRNA (in which the 3′-end of 5.8S and 5′-end of 25S rRNA are linked by ITS2) and some 7S pre-rRNA (in which the 3′-end of 5.8S rRNA is extended) are also recovered. This indicates that C2 cleavage, which generates the 3′-end of the 7S pre-rRNA, has occurred in most Arx1-associated particles, but processing of 7S to the 5.8S rRNA has not yet taken place in all particles26
(data not shown). This is in agreement with the reported cytoplasmic location of the final step in 5.8S rRNA processing46
. The foot
structure may therefore represent retained factors involved in processing and/or folding of ITS2 or it might protect this region and/or offer a binding platform for processing factors until nuclear export and/or maturation of this area are completed.
The position of the unconventional export receptor Arx1 could be unambiguously assigned to the characteristic knob
structure observed on either the Rix1- or Arx1-purified 60S subunit particle. The knob
structure is located directly in front of the exit tunnel and the observed binding site of Arx1 is in good agreement with an earlier suggestion that it may bind in close proximity to Rpl2543
. The flexible rRNA expansion segment ES27 was suggested to be involved in regulating the access of non-ribosomal factors to the exit tunnel36
. Binding of Arx1 to the pre-ribosome at the exit tunnel and interaction with ES27 might favor the ES27out
conformation, which potentially facilitates nuclear export of the subunit. Arx1 is structurally related to methionine aminopeptidases (Met-APs), which remove N-terminal methionine from the nascent polypeptide chain; however, Arx1 lacks this enzymatic activity20
. It is therefore plausible that Arx1 binds the pre-60S subunit in a similar manner and position to Met-AP on the mature 60S subunit. We speculate that Arx1 could function as a placeholder for Met-AP and/or other cytoplasmic translation-associated factors that bind to the exit tunnel. In this way, Arx1 could restrict the access of such factors to the exit tunnel region of the immature 60S subunit. This could avoid steric hindrance during export of the 60S subunit (note that Arx1 also acts as export factor; see below) or premature association of translation-competence. Last but not least, Arx1 may act as quality control factor to ensure correct assembly of this key site on the 60S subunit before the particle is exported to the cytoplasm.
Finally, the localization of the export factor Arx1 supports the model that transport receptors are distributed over different regions of the pre-60S subunit. The export adapter Nmd3 was found to interact with the intersubunit surface of the mature 60S subunit17
. Mex67-Mtr2 was shown to interact with 5S rRNA in vitro18
and may thus bind to the central protuberance at the “top” of the subunit. Arx1 binds close to the exit tunnel on the opposite side of the pre-60S subunit, a location that is very distant from the predicted interaction sites of the other two export factors. It might have been envisaged that nuclear export factors would be needed to initiate subunit export and then “tow” the subunit through the nuclear pore complex. However, the dispersed distribution of export factors on the pre-60S surface supports a view that no single region of the 60S subunit is sufficient for efficient passage through the nuclear pore. Rather, several export factors scattered over the complete surface of the pre-60S subunit are required to shield and efficiently export this huge cargo through the hydrophobic FG repeat phase of the nuclear pore into the cytoplasm.