Distinct roles of nuclear basket proteins in directing the passage of mRNA through the nuclear pore

Abstract

The in vivo characterization of the exact copy number and the specific function of each composite protein within the nuclear pore complex (NPC) remains both desirable and challenging. Through the implementation of live-cell high-speed super-resolution single-molecule microscopy, we first quantified the native copies of nuclear basket (BSK) proteins (Nup153, Nup50, and Tpr) prior to knocking them down in a highly specific manner via an auxin-inducible degron strategy. Second, we determined the specific roles that BSK proteins play in the nuclear export kinetics of model messenger RNA (mRNA) substrates. Finally, the three-dimensional (3D) nuclear export routes of these mRNA substrates through native NPCs in the absence of specific BSK proteins were obtained and further validated via postlocalization computational simulations. We found that these BSK proteins possess the stoichiometric ratio of 1:1:1 and play distinct roles in the nuclear export of mRNAs within live cells. The absence of Tpr from the NPC predominantly reduces the probability of nuclear mRNAs entering the NPC for export. Complete depletion of Nup153 and Nup50 results in an mRNA nuclear export efficiency decrease of approximately four folds. mRNAs can gain their maximum successful export efficiency as the copy number of Nup153 increased from zero to only half the full complement natively within the NPC. Lastly, the absence of Tpr or Nup153 seems to alter the 3D export routes of mRNAs as they pass through the NPC. However, the removal of Nup50 alone has almost no impact upon mRNA export route and kinetics.

Function of the Nuclear Transport Machinery in Maintaining the Distinctive Compositions of the Nucleus and Cytoplasm

“…The structure of the annular scaffold is conveniently thought of as a central annular core sandwiched between cytoplasmic (“outer”) and nucleoplasmic (“inner”) rings ( Figure 1 A) and complementary studies using cryo-EM and X-ray crystallography have developed detailed models of the way in which the chains of the various nuclear pore proteins (nucleoporins or “nups”) are arranged in these components and how the pores are attached to the nuclear envelope membrane [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 ]. Fibrous appendages project from both the cytoplasmic and nucleoplasmic faces of the nuclear pores ( Figure 1 A), with those in the nucleus contributing to the formation of a distinctive nuclear basket [ 11 ]. These fibrous appendages function as binding sites for a range of factors involved in both transport and gene expression [ 12 , 13 ].…”

Spelling out the roles of individual nucleoporins in nuclear export of mRNA

“…Notably, the interaction between FG-Nups and transport receptors does not appear to confer directionality upon the cargo, as is evidenced by similarity of interaction between import and export interactions between transport receptors and FG-Nups [ 126 , 146 , 147 ]. Upon passing through the FG-rich central channel of the NPC, mRNPs then dissociate from cytoplasmic fibril Nup214 via essential mRNP export factors Gle1, IP6, and DDX [ 35 , 129 , 148–152 ] and difuse into the cytoplasm.…”

“…Using a combination of Single-Point Edge-Excitation sub-Diffraction (SPEED) microscopy and Auxin Inducible Degron (AID) technology, a technology that enables the rapid and highly specific degradation of target Nups, a pair of recent publications have made great strides in unraveling the relationship between basket Nups and the docking and export of mRNA [ 83 , 129 ]. The utilization of these two techniques in concert has revealed that basket Nups play a much larger role in nuclear docking and export than previously thought.…”

“…In another study focused on the dynamics of mRNA export in the absence of targeted Basket Nups, Li and colleagues employed AID strategies to observe the alterations in export behavior or mRNA via SPEED microscopy [ 129 ]. SPEED microscopy is a super-resolution technique permitting a spatial localization precision during in vivo imaging of approximately 10 nm and a temporal resolution up to 0.4 ms that has been employed to great effect to track macromolecules as they move through the NPC or primary cilium of the cell [ 144 , 163–167 ].…”

“…In fact, many mRNPs will fail to export and then be re-imported into the nucleoplasm. This allows researchers to evaluate the regional exportive failure due to the absence of a specific basket Nup as well as the total export efficiency, which is defined as the number of successful export events divided by the total number of successful and abortive events [ 129 ].

Figure 2.

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