Passage Review: miRNAs and the transmission of genetic information to proteins, Part I

Let’s jump into another passage. If you want a primer on how to tackle passages well, please take a look at my 3-part “dummies” series first: Part I, Part II, and Part III.

It’s low-key crucial to review that series first before diving in, because as you’ll see, not all passages are created equally. Some are nearly impenetrable, technical articles, while others are merely tarted up reviews of a concept that resemble something hastily scraped from a college textbook. You’ll need to be comfortable constructing a passage map, because that’s the only way to guarantee you’ll be able to answer every question for that passage correctly.

Here’s paragraph 1 of the passage we will be going over today.

If anyone has an extraordinarily concise summary of this paragraph, please post it as a comment below!

During the MCAT, I’m way too wired from pure adrenaline, so I find my thoughts tend to wander too frequently while reading a passage. But when I draw pictures, the relationships come to life. Remember from Part I:

Tip 3: Always, always, always draw a picture

How do you know what’s important? Everything is. So understand every word. Here’s how I summarize above:

Paragraph 1:

Paragraph 1 illustrated for my passage map

Isn’t this much friendlier than pure text? We see miRNA hybridizing with our mRNA in the bottom, driving home the point of miRNA’s regulating expression by modifying your mRNA in a manner to thwart translation. After all, how could our rRNA proceed with translation with an obstacle in its tracks, the miRNA? Notice how I put an asterisk over the arrow pointing from DNA to miRNA. The reason is because there are actually two ways for miRNA to arise, according to the passage, and they each follow distinct paths, noted by the dashed arrows, to form the miRNA / miRNA* duplex.

Also, direct your attention to the conditionals on the bottom left of Paragraph 1’s illustration. Notice how we have silencing accomplished in two distinct ways, one via translational repression (when there’s partial complementarity) and the other via endonucleolytic cleavage. I don’t claim to be an expert on either; all I know is that they’re different avenues of silencing.

The rest of the passage looks like this, for a total of 4 paragraphs and 2 figures:

Paragraph 2:

PDAC miRNA poorly understood

Not much to report here, other than the written equivalent of a throat-clear introducing the experiment’s motivation

Paragraph 3:

I’m going to slow down for this one because this is an example of how the Altius method of creating relationships

EMT ↑ ⟼ metastasis ↑

EMT cells: adherent, polar ↓ ⟼ migratory ↑ = metastatic

Remember how we need to remember every passage detail, otherwise there’s a chance we will have to rely on our previously-accumulated knowledge to deduce the answer rather than expertly tease the answer out from the wealth of information actually presented in the passage. Low-key, the MCAT will throw several pieces of evidence for the correct answer, as you’ve probably noticed while studying. Also, check out the first problem associated with this passage a little ways below to see a perfect example of the MCAT throwing you multiple pieces of evidence to guide you towards the correct answer. It’s good for several reasons, but my favorite is that it gives conscientious test takers a chance to confirm their answer.

miR-200 ↑ ⟼ TFs ZEB1/2 ↓ ⟼ EMT↓

The above relationships should be pretty evident, but let’s slow down for E-cadherin, which the passage posits as responsible for regulating EMT. But what arrow do we use? Well, the passage continues by noting that E-cadherins maintain cell polarity and have to do with adhesion. From our second relationship above, we can clearly see how E-cadherins encourages cells to stay on the E side of the EMT — that is, the side where cells are adherent and polar. So now we can note:

E-cad ↑ ⟼ adherent, polar ↑ ⟼ EMT↓

and we can actually expand this relationship given our first relationship for Paragraph 3:

E-cad ↑ ⟼ adherent, polar ↑ ⟼ EMT↓ ⟼ metastasis ↓

The passage then explains the point of the experiment:

MI: does miR-200 ↑ ⟼ Epithelial ↑?

Leading us to the first set of graphs:

Figure 1:

Remember the mnemonic, DRY MIX: the Dependent variable, or the Response variable, goes on the Y-axis, while the Manipulated variable, also known as the Independent variable, gets plotted on the X-axis. It’s really important to keep these straight because we will be relying on our R-squared value— a goodness-of-fit measure for linear regression models — to quantitatively gauge how much variance in the dependent variable is explained by the variance in the independent variable.

Our x-axis in this case is quantity of miR-200b, which we see gets manipulated in the sense that we gradually throttled up the concentration from 0 to 3.5 for E-cadherins and from 0 to 100 for the transcription factors, ZEB1/2.

top two plots of Fig. 1

We can quickly note that the top two plots in Fig 1. can be summarized as follows:

E-cadherin: sum / mean miR-200b ↑ ⟼ E-cad ↑

This is justified because the p-value is very small (7.5e-5 and 1.3e-4), indicating that we are dealing with a statistically significant positive correlation.

Let’s move onto ZEB1:

Here we see a statistically significant (as per the p-value) but weak (per the R-squared) negative correlation. So we can note that

miR-200b ↑ ⟼ ZEB1 ↓ [WEAK]

And finally, ZEB2:

Here, an R-squared value of 0.15 can be interpreted as almost no correlation. In fact, we can summarize that observation as

miR-200b ↑ ⇏ ZEB2

Cross out your “implies” arrow to denote how there’s no relationship we can definitively establish between our miRNA and ZEB2.

Almost done:

Paragraph 4:

Prostate CA ↑ ⟼ miR-34a ↓

Note: CA == cancer

miR-34a = E-cad

We can simply summarize miR-34a as being equivalent to E-cad, which means the relationship we noted earlier can be applied to miR-34a:

miR-34a = E-cad ↑ ⟼ adherent, polar ↑ ⟼ EMT↓ ⟼ metastasis ↓

The researchers rehabilitated the miR-34a content in prostate cancer cells via recombinant therapy, which means using a vector to introduce novel genes.

Figure 2:

So when reintroduce miR-34a expression, our cell culture doesn’t grow in size as quickly as our vector-only PDAC group.

Line 1: miR-34a ↑ ⟼ growth ↓

Line 2: miR-34a ↑ ⟼ growth ↓↓

Here I should note that the size shrinks relative to the control — the vector only.

In summary:

Paragraph 1:

Paragraph 1 illustrated for my passage map

Paragraph 2:

PDAC miRNA poorly understood

Paragraph 3:

EMT ↑ ⟼ metastasis ↑

EMT cells: adherent, polar ↓ ⟼ migratory ↑ = metastatic

miR-200 ↑ ⟼ TFs ZEB1/2 ↓ ⟼ EMT↓

E-cad ↑ ⟼ adherent, polar ↑ ⟼ EMT↓ ⟼ metastasis ↓

MI: does miR-200 ↑ ⟼ Epithelial ↑?

Figure 1:

E-cadherin: sum / mean miR-200b ↑ ⟼ E-cad ↑

miR-200b ↑ ⟼ ZEB1 ↓ [WEAK]

miR-200b ↑ ⇏ ZEB2

Paragraph 4:

Prostate CA ↑ ⟼ miR-34a ↓

miR-34a = E-cad ↑ ⟼ adherent, polar ↑ ⟼ EMT↓ ⟼ metastasis ↓

Figure 2:

Line 1: miR-34a ↑ ⟼ growth ↓

Line 2: miR-34a ↑ ⟼ growth ↓↓

And now, we are ready to answer any question this passage throws our way. That’ll be detailed in Part II of this series. But if you’re impatient, here’s a little teaser.

What’s the first thing we do? Check out Part I of my introductory series on analyzing passages if you need a refresher. We have to restate the question stem. OK, let’s read it one word at a time: some miRNA-mRNA interactions lead to disease, so a new therapy is using anti-miR to silence these gene silencers. Recall how we illustrated the miRNA-mRNA interaction:

Now the idea is to find an anti-miR(NA) to pair up with the miRNA to prevent the miRNA from pairing up with the mRNA. We are given the mRNA sequence and asked to find the most probable anti-miRNA sequence that would stymie mRNA-miRNA interactions.

Before I move forward, let’s recall some of our transcription nomenclature. Bear with me here.

Why do we call the strand that is NOT transcribed the coding or sense strand? Because if you convert the coding / sense / anti-template DNA strand into RNA, you’d be able to manufacture proteins (hence why we call sense DNA positive DNA, since the RNA version of sense DNA is a valid transcript for direct translation). Hence, the sense mRNA strand has the same sequence as the coding / sense strand (with U’s for T’s and 2' hydroxyls along the single-strand), and that mRNA strand is complementary to the template, or antisense, or anti-coding strand. Sense mRNA and sense DNA sequences are mutually complementary to that template strand.

Why did I explain that in excruciating detail? Let’s suppose I gave you a problem where I gave you the template strand sequence and asked you to determine which of the following four sequences represents a valid complement to the sense mRNA synthesized from that template strand sequence. But wait a tick: if sense mRNA is complementary to the template strand sequence and we are asked to find a sequence complementary to that mRNA transcript, then do we even need to convert the template strand sequence into mRNA and then find the complement of that sequence? Definitely not. The complement to the complement to a sequence is the original sequence.

So back to the problem. We are asked to find an anti-miR sequence that would interact with the miRNA that would otherwise interact with the mRNA sequence presented in the question stem. But as that extended analogy just showed, we already know what sequence we need to interact with the miRNA: it’s already given!

This is absolutely amazing, because now our restated question stem completely trivializes the problem. All we have to ask for each answer choice is, “the following subsequence is contained in 5'-CUA..UGU-3':”

Does that make sense? We eliminated all the BS by thinking really hard about the crux of the question so we can quickly verify our Frankenstein sentences we are about to make (see Part I, Part II, and Part III if that phrase is unfamiliar to you). Let’s concatenate our restated question stem with each answer choice and evaluate them one at a time.

A. the following subsequence is contained in 5'-CUA..UGU-3': 5'-UGCUGCUGA-3'

By inspection, we see that:

Choice A is the second row

So, yes! Option A is a valid subsequence of the mRNA transcript.

The power of the Altius method is the ability to zero in on the correct answer quickly. You’re now gifted with additional time to prove to yourself that this is, indeed, the correct answer. It’s at the 3'-UTR, as well, and we see complete complementarity for 7 consecutive bases — 2 through 8 — starting from the 3' end. This matches our conditionals in Paragraph 1 illustration. A is a great one to keep as a definitely maybe.

Let’s try the rest of the answers.

B. the following subsequence is contained in 5'-CUA..UGU-3': 5'-ACAGCAGCA-3'

So many of you probably scan in chunks like this, where you compare the sequence of the answer choice to the sequence in the question stem:

We don’t see a match. So we move on:

Nothing.

Nope. But what about complementarity? Complements are anti-parallel, so we can’t do a direct comparison. We need to flip it:

The sequence is complementary to the mRNA transcript. This means that it would not bind to the miRNA that would silence the mRNA transcript. In fact, this a legitimate sequence for the miRNA itself, and therefore would not be a valid therapy if the intent was to interact with miRNA to prevent silencing. Hence, we reject B.

C. the following subsequence is contained in 5'-CUA..UGU-3': 5'-ACAGCAGCA-3'

Let’s scan across the mRNA transcript again:

So let’s move forward

Interesting:

So, we have another contender for the correct answer right? Choice C ostensibly appears to be a valid subsequence of the mRNA transcript.

Right?

Well, the passage devoted considerable real estate to making a rather specific distinction between partial complementarity and complete complementarity. We needed exactly 7 for complete recognition, and if we look at the choice A box (red), we see we have an overlap of 8 bases. Sufficient for the criteria established in our passage map. For C, on the other hand, we only have 5 of the required 7 consecutive bases. In other words, our anti-mR will be unable to recognize our miRNA to silence in the first place, so this is a therapeutically useless sequence. Eliminate C.

We are almost there.

D. the following subsequence is contained in 5'-CUA..UGU-3': 5'-CGCGCCGGGG-3'

Let’s do our analysis:

Moving forward:

OK, let’s stop. We can’t just ignore that quadruple G. That’s complementary to the mRNA quadruple C. See?

Now we can quickly reject D for the same reason we eliminated B.

And that’s it for our preview, ladies and gents. I appreciate your attention. Stay tuned for more questions that illustrate the power of the Altius method in tackling MCAT passages.

UPDATE: Part II is available here!