Problem-solving test: Nucleocytoplasmic shuttling of pre-mrna binding proteins



Terms to be familiar with before you start to solve the test: transcription, pre-mRNA, RNA processing, RNA transport, RNA polymerase II, direct and indirect immunofluorescence staining, cell fractionation by centrifugation, oligo(dT)-cellulose chromatography, washing and elution of the column, ribonuclease, SDS-polyacrylamide gel electrophoresis, Western-blotting, pre-ribosomal particles, nuclear envelope, heterokaryon, phase contrast microscopy, polyadenylated RNA.


Newly synthesized transcripts of protein-coding genes (pre-mRNAs) of eukaryotic cells bind dozens of nuclear proteins forming large ribonucleoprotein complexes called pre-mRNPs. Proteins of these complexes are involved in the processing and transport of pre-mRNAs. The experiments described in this test deal with the nucleocytoplasmic distribution of two of these proteins, protein A1 and C [1].

A human tumor cell line (HeLa cells) was cultured in the absence (Figs. 1 A a and b) or presence of actinomycin D (Act D)1 (c and d), an inhibitor of RNA synthesis, for 3 hours (the concentration used was sufficient to block transcription by RNA polymerase II). The cells were fixed and immunostained using anti-C (charts a and c) or anti-A1 antibodies (b and d), according to an indirect immunofluorescence cytochemistry protocol.

In the experiment of Fig. 1 B, HeLa cells were incubated in the absence (lanes 1, 2, and 5) or in the presence of actinomycin D (lanes 3, 4, and 6) for 3 hours, and then exposed to ultraviolet (UV) irradiation (lanes 1 to 4). Nonirradiated cells (lanes 5 and 6) were used as controls. UV-irradiated cells were fractionated into nuclear and cytoplasmic fractions by centrifugation. Nuclear (lanes 1 and 3), cytoplasmic (lanes 2 and 4), and total cellular fractions (lanes 5 and 6) were subjected to affinity chromatography on separate oligo(dT)-cellulose columns. Loosely bound cellular component were removed by extensive washing and then oligo(dT)-bound materials were eluted from the columns. Eluted samples were digested by ribonuclease and fractionated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Western blot analysis was performed using anti-A1 and anti-C antibodies.

Figure 1.

The effect of actinomycin D (Act D) on the cellular distribution of protein A1 and C. A. Immunofluorescence cytochemical visualization of the proteins. B. Identification of protein A1 and C in oligo(dT)-cellulose-bound complexes of control and UV-irradiated cellular extracts (for experimental details see the text); the vertical arrow indicates the direction of electrophoresis).

1 The abbreviations used are: Act D, actinomycin D; CHX, cycloheximide; MCQ, multiple-choice question; SDS, sodium dodecyl sulfate; UV, ultraviolet.


This type of question consists of a question or incomplete statement followed by five suggested answers or completions. Select the one best answer.

  • 1____ Based on the experimental protocol described above, determine the aim of oligo(dT)-cellulose chromatography in this study:
    • A: To isolate pre-rRNA and rRNA

    • B: To isolate pre-ribosomal RNP particles

    • C: To isolate pre-mRNA and mRNA

    • D: To isolate pre-mRNP and mRNP

    • E: C and D

  • 2____ Based on the experimental protocol and the results presented in Fig. 1 B, determine the aim of UV-irradiation:
    • A: To inhibit transcription by RNA polymerase II

    • B: To disrupt the nuclear envelope

    • C: To dissociate proteins from RNA molecules

    • D: To cross-link RNA-bound proteins to the RNA molecules

    • E: None of the above

  • 3____ What kind of Western-blot images would have been produced without prior RNase treatment of the samples?
    • A: No bands would have appeared in the samples

    • B: Bands of protein C and A1 would have appeared in UV-untreated samples (5 and 6) only

    • C: Protein C and A1 would have migrated slower

    • D: Protein C and A1 would have migrated faster

    • E: RNase treatment would have not altered the immunoblot pattern

  • 4____ What conclusions can be drawn by comparing sample 1 and 2 in Fig. 1 B?
    • A: Protein C binds to pre-mRNA

    • B: Protein A1 binds to pre-mRNA

    • C: Protein C and A1 are present in the nuclei of HeLa cells

    • D: A and B

    • E: A, B, and C

  • 5____ What conclusions can be drawn by comparing sample 1 and 5?
    • A: UV-irradiation induces the synthesis of protein C and A1

    • B: C and A1 bind to pre-mRNA by covalent bonds in untreated cells

    • C: C and A1 bind to pre-mRNA by noncovalent bonds in untreated cells

    • D: A and B

    • E: A and C

  • 6____ What conclusions can be drawn by comparing sample 2 and 4?
    • A: mRNAs disappeared from actinomycin D-treated cells

    • B: UV-irradiation inhibited the synthesis of protein C

    • C: mRNA-bound protein A1 accumulated in the cytoplasm of actinomycin D-treated cells

    • D: A and B

    • E: A, B, and C

Figure 2 shows the results of an experiment using interspecies heterokaryons. HeLa cells were fused with cells from the frog Xenopus laevis. Heterokaryon cultures were left untreated (charts a to c, Fig. 2) or treated with the protein synthesis inhibitor cycloheximide (CHX; d to f). The cultures were double-stained with rhodamine-labeled anti-A1 (a and d) and fluorescein-labeled anti-C antibodies (b and e) specific for the human proteins (rhodamine and fluorescein are fluorescent dyes of different color). Cultures were also visualized by phase contrast microscopy (c and f).

Figure 2.

Localization of proteins A1 and C in heterokaryons of HeLa and X. laevis cells (for details see the text; arrows indicate X. laevis nuclei).

Study the protocol and results of the experiments and solve the following multiple-choice questions (MCQs).


The following statements are related to the information presented in the description of the experiment. Based on the information given, select

  • A: if the statement is supported by the information given.

  • B: if the statement is contradicted by the information given.

  • C: if the statement is neither supported nor contradicted by the information given.

  • 7____ Protein C binds to pre-rRNA.
  • 8____ Protein A1 binds to mature rRNAs.
  • 9____ Actinomycin D treatment makes the nuclear envelope leaky for proteins.
  • 10____ UV-irradiation stimulates the nucleocytoplasmic export of proteins.
  • 11____ Nucleocytoplasmic export of protein A1 takes place in actinomycin D-treated cells only.


In this type of question a set of lettered headings is followed by a list of numbered words or phrases, select

  • A: if the word or phrase is associated with A only.

  • B: if the word or phrase is associated with B only.

  • C: if the word or phrase is associated with A and B.

  • D: if the word or phrase is associated with neither A nor B.

  • A: Protein A1

  • B: Protein C

  • C: Both of them

  • D: Neither of them

  • 12____ Is imported into the nuclei of HeLa cells.
  • 13____ Can be exported from the nucleus to the cytoplasm.
  • 14____ Is able to bind to mature mRNA.
  • 15____ Cycloheximide treatment stimulates its nucleocytoplasmic transport.
  • 16____ Is taken up by X. laevis nuclei in the heterokaryons.


This type of question consists of a question or incomplete statement followed by five suggested answers or completions. Select the one best answer.

  • 17____ What are the main conclusions drawn from the experiments?
    • A: Protein A1 shuttles between the nucleus and cytoplasm of cells

    • B: Protein C is a resident nuclear protein

    • C: Both proteins are able to bind to polyadenylated RNA molecules

    • D: Both proteins may be involved in the cellular metabolism of pre-mRNA

    • E: All of the above


  • 1D
  • 2D
  • 3C
  • 4E
  • 5C
  • 6C
  • 7C
  • 8C
  • 9B
  • 10C
  • 11B
  • 12C
  • 13A
  • 14A
  • 15D
  • 16A
  • 17E


Protein A1 and C were isolated from HeLa cell fractions by oligo(dT)-cellulose chromatography; both of them are thus able to bind to polyadenylated RNA species, pre-mRNA and mRNA (MCQ 1: D; MCQ 4: E; MCQ 7: C; MCQ 8: C; MCQ 14: A). This binding is stabilized by UV-irradiation indicating that UV light induces covalent crosslinking of RNA and proteins bound to it (MCQ 2: D) and that without this crosslinking, noncovalently bound proteins are easily released from the RNAs under denaturing conditions (MCQ 5: C). RNase treatment served to release protein A1 and C from the RNA molecules (MCQ 3: C).

The experiments indicate that both protein A1 and C are predominantly nuclear (MCQ 12: C). Although protein C is a resident nuclear protein, protein A1 can be exported to the cytoplasm (MCQ 13: A) and then reimported into the nucleus (MCQ 16: A). Export of protein A1 is selectively stimulated by actinomycin D treatment (MCQ 6: C; MCQ 9: B) but happens in untreated heterokaryons as well (MCQ 11: B). The uptake of human A1 protein by X. laevis nuclei is not affected by simultaneous cycloheximide treatment indicating that it is not newly synthesized A1 but A1 molecules already present in HeLa nuclei that are translocated to the frog nuclei (MCQ 15:D). The experiments do not provide information about the effect of UV-irradiation on nuclear protein transport (MCQ 10: C).