Discovery Talk Questions and Answers: SNURPs and Serendipity

Joan Steitz
Assessments created by Dr. Kassandra Ori-McKenney

Quesitons

In her talk, Dr. Steitz describes splicing. What is splicing?
1. The removal of non-coding DNA from pre-mRNA.
2. The removal of non-coding RNA from pre-mRNA.
3. The removal of proteins from pre-mRNA.
4. None of the above.
In the relevant paper, the authors study the splicing of adenovirus mRNA species. How are adenoviral early RNA sequences spliced?
1. By the host cell’s splicing machinery.
2. By adenoviral splicing machinery.
3. By the host cell’s ribonucleases.
4. All of the above.
In her talk, Dr. Steitz said that she wanted to make antibodies that would recognize the components of the machinery responsible for splicing.
How did she obtain these antibodies?
In her talk, Dr. Steitz mentions the second piece of serendipity was the use of Protein A. What did this new reagent enable her and her lab to do?
In their paper, the authors find that after HeLa cell infection with adenovirus, the major products of transcription of the adenoviral genome are correctly spliced RNA molecules. What happened when they incubated the nuclear extract with antibodies from SLE patients?
For one experiment in their paper, the authors use a mercurated nucleotide, Hg-CTP, and chromatography in order to:
1. Slow the maturation process of the RNA species.
2. Alter the amount of adenoviral RNA species that were synthesized.
3. Separate newly synthesized from endogenous RNA.
4. None of the above.
1. In their paper, why did the authors look at the effect of the SLE antibodies on polyadenylation?
2. What was the outcome of this experiment?
Which statements accurately describe the anti-Sm and anti-RNP antibodies? (Select all that apply).
1. Both antibodies recognize U1 snRNA species.
2. Both antibodies recognize only RNA components of the snRNP.
3. Neither antibody recognizes the U6 snRNA species.
4. The antigen(s) recognized by both antibodies is necessary for splicing.
5. None of the above
In the paper, it is noted that “these antibodies do not inhibit all the splicing events we have studied with equal efficiency.” Provide a possible explanation for this observation.
Although humans contain a similar number of genes as the fruit fly, humans are much more complicated. How might splicing contribute to this?

Answers

b; The removal of non-coding RNA from pre-mRNA.Pre-mRNA contains both introns (non-coding RNA regions) and exons (coding RNA regions), but in order for it to mature into an mRNA, the intron regions are first spliced out.
(03:43): “Evidence came together from laboratories from all over the world conclusively convinced everyone that our genes, in fact, were not like bacterial genes but contained segments of nonsense, the so-called introns. And that these introns were then spliced out once the entire gene was made into pre-messenger RNA and that was what enabled the cell to send a fully formed message to the cytoplasm to be translated by the ribosomes.”
a; By the host cell’s splicing machinery.
p. 1371: “It therefore seems reasonable to infer that adenoviral early RNA sequences are spliced by the host cell’s splicing machinery.”
AND

p. 1373: “adenoviral late RNA sequences are polyadenylylated and spliced correctly in isolated nuclei.”
She was not able to synthesize these antibodies because the nuclear RNA binding proteins are non-immunogenic. Instead, she found that patients with certain autoimmune diseases (MCTD and lupus) made antibodies against certain nuclear RNA protein complexes, so she purified these antibodies from the patients.Video (03:19): “So, for half a year, I tried to make antibodies against these nuclear RNA binding proteins but this was without success because these are very highly conserved proteins and therefore very non-immunogenic.”
Video (05:09): “Several people had said that they thought they’d heard of some diseases where patients made auto-antibodies, antibodies against their own cellular components, that reacted with nuclear RNA protein complexes.”

Video (06:15): “(…) the auto-antibodies would then recognize the cellular component and we would use them as tools to both purify and to characterize the function of the target of the auto-antibodies.”

p. 1371, Materials and Methods section: “(. . .) preincubation (. . .) in the presence of IgG prepared from sera of SLE patients.”
They used the Protein A beads and the antibody sera from different patients with autoimmune disorders to immunoprecipitate the RNAs and proteins that composed the small nuclear ribonucleoprotein particles. They were therefore able to identify the RNAs and proteins that were in these particles.(08:02): “Michael’s own serum, which was used as a control in the next lane immunoprecipitated no RNAs, but what you see on the right-hand side here, is the pattern that he obtained with various different auto-antibodies from various patients. And for the first time, we could see what the RNAs were, and then we could also use this to find out what the proteins were, in these little particles. So, what we now know, is that these very highly conserved and very abundant targets of the auto-antibodies are, in fact, the SNURPs.”
The antibodies inhibited the splicing of the adenoviral pre-mRNA species.p. 1373: “We may therefore conclude that anti-RNP and anti-Sm antibodies specifically inhibit the synthesis in isolated nuclei of RNA chains that correspond in size to fully matured mRNA species.”
p. 1373: “Anti-RNP antibodies inhibit splicing of adenoviral early RNA.”

p. 1374: “. . . that adenoviral early RNA molecules are spliced correctly in isolated nuclei and that anti-RNP inhibits the appearance of such spliced products.”
c; Separate newly synthesized from endogenous RNA.p. 1373: “The mercurated RNA chains made in vitro were purified by chromatography on thiol-agarose columns, under stringent conditions that permit a complete separation of newly synthesized from endogenous RNA.”
a. Because they wanted to rule out the possibility that the SLE antibodies were inhibiting splicing by disrupting the polyadenylation of the RNA.p. 1374: “During the maturation of both early (31) and late (32) adenoviral RNA, polyadenylylation of 3’termini appears to precede splicing. It is therefore possible that the inhibition of synthesis of spliced adenoviral RNA species observed in these experiments is a secondary consequence of a disruption in polyadenylylation.”
b. They found that polyadenylation was not dramatically decreased in the presence of the SLE antibodies, so the SLE antibodies most likely inhibited splicing.

p. 1374: “The results presented in Table2 reveal that neither the overall polyadenylylation of RNA sequences labeled in isolated nuclei nor the addition of poly(A) to viral sequences is much affected by incubation of nuclei with anti-RNP antibodies.”
a and d.b. is FALSE and c. is FALSEFor d.
p. 1374, Discussion: “We therefore conclude that an antigen(s) recognized by both anti-RNP and anti-Sm is necessary not only to the splicing reactions we have studied but almost certainly to those that mature cellular mRNA species.”

For a., b., and c.
p. 1374, Discussion: “Anti-Sm immunoprecipitates snRNPs containing five different HeLa cell snRNA species, U1, U2, and U4-U6, whereas anti-RNP recognizes only structures containing U1 RNA (12). A protein component of the snRNP appears to be recognized by anti-Sm, whereas anti-RNP is directed against a determinant that consists of both RNA and protein (35-37).”
The cellular splicing machinery could be modified or replaced with viral components as the infection progresses.p. 1375: “Although there are a number of possible explanations for this observation, it does raise the intriguing possibility that the cellular splicing machinery becomes partially modified or wholly replaced by viral components as the infection progresses.”
Due to splicing, the same gene can code for (or make) multiple proteins.
For example, the same pre-mRNA can have its introns spliced out, or can have its intron and an exon spliced out, leading to two different mRNA products that are translated into two different protein products.

Note: This question is designed to make students think about how splicing can make multiple gene products, but it is also mentioned in both the paper and Dr. Steitz’s talk.

p. 1372-1373: “the chains of 550 and 880 nucleotides correspond in size to two mature (spliced) mRNA species complementary to early region 1A.”

Video (10:01): “And also, as we’ve understood about the spicing process, we know that splicing is the reason why we can have the same number of genes in our genome as the fruit fly Drosophila and yet be more complicated. And this is because we do splicing in alternative ways, therefore, making the most out of every gene, making multiple products from every gene.”