Transcription and Translation in Mammalian Cells. 00:34:07.21 which is important for RNA localization -- actin/RNA localization -- 00:31:14.11 I mean from modifying the mating type switching. 00:36:18.02 And about half an hour later, they've all gone back to the state which we call the occult state, 00:02:09.09 Just to refresh your memory, what is the central dogma that allows us to look at these things 00:36:24.16 where they cannot be accessed by the probe. 00:03:49.09 what genetic sequence that corresponds to. 00:28:26.25 And you can see RNAs move around a lot in these neurons.

00:00:35.08 toa ctually image gene expression within single cells. 00:32:57.10 It has no choice. 00:36:53.12 until the neuron's been stimulated. 00:21:03.15 until they find a spot on the pore to dock and to be processed for export. 00:33:44.13 to its end point. 00:27:51.25 junk put on its essential gene, not only the MS2, but also the capsid proteins. 00:33:36.08 They block the translation of the RNA so that the RNA can then move, unimpeded by ribosomes, © 2020 Albert Einstein College of Medicine |. 00:16:03.13 And using these three colors, you can then determine from the transcription sites, 00:36:33.13 come right back. 00:29:17.23 And this... when this was published, this was actually put on YouTube under the heading, 00:30:58.24 And the protein that's gone into the daughter nucleus now effects a change in the genotype 00:23:53.16 RNA to be sorted. 00:31:30.09 In the... in the yeast to the left, you see that it's only in the daughter cell. 00:19:18.11 And then you can take that and go back, now, to the tissue with sequences that you're interested in. In Situ Hybridization of Yeast Cells (RNA and oligonucleotide probes) Single Cell Gene Expression Profiling: Multiplexed Expression Fluorescence in situ Hybridization (FISH) Application to the Analysis of Cultured Cells ; Contact Robert Singer with suggestions, comments or questions. 00:20:13.20 for a pore that's gonna let it out. 00:23:11.22 So, let's take a look at this process. 00:17:58.13 the different gene expressions are... are described in the nucleus. 1994 Dec 22-29;372(6508):727-8. 00:02:03.18 And they phosphorylate the proteins that are repressing the RNA from being translated. 00:23:58.20 So, the previous MS2 system didn't degrade rapidly, and therefore it messed up

Singer begins by explaining that cell cultures are heterogeneous and analyzing single cells provides spatial and temporal information not available from bulk analysis. Robert Singer is a professor at The Albert Einstein College of Medicine in the Departments of Anatomy and Structural Biology, Cell Biology, and Neuroscience, and co-director of the Gruss Lipper Biophotonics Center. 00:22:20.11 So, how does this occur?

00:11:40.11 not a directed event. 00:27:52.03 and gets modified.

00:16:23.09 transcription site, you can build a picture of how many polymerases are on this gene,

Nerve Growth Factor Production by Muscle Cells. 00:02:55.17 a high numerical aperture objective so you can collect as much light as you can Area of Research: We study single mRNA molecules from cradle to grave in single living cells using high resolution imaging. 00:16:55.12 at the end, indicating that termination of the transcript is a rate-limiting step 00:09:37.28 But as soon as they're out in the cytoplasm, you see red RNAs. 00:22:39.00 localized to the bud tip and makes the protein, here, that goes into the daughter nucleus. 00:34:17.16 Because if you look in knockout mice, the RNA cannot localize at all here. 00:17:03.13 in making the RNA, the mature RNA that goes into the cytoplasm. 00:02:59.20 Well... so, what does that mean? Correlation between transcripts from two alleles of a constitutively active gene, MDN1, in diploid cells. 00:31:36.04 And then, in this 20-minute time course, within 10 minutes RNA shows up at that site. 00:17:46.25 Here are the genes that we looked at. 00:02:42.17 And we're going to see those events occur in cells by looking at these individual molecules. 00:38:01.18 You get a stimulation, you activate, you strengthen the synapse. 00:17:37.11 Now, remember, we have a pore which is several hundred microns... nanometers in width. 00:17:27.16 and some are green and red, and so forth. 00:38:54.09 or is stimulated in some way. 00:22:32.17 floating around randomly. 00:20:42.26 that when they hybridize together on the same RNA they allow you to generate an... 00:32:33.06 And finally, if you just leave out the uncaged neurotransmitter, and then... but use the laser, In addition, these techniques can be used to unveil the dynamics of RNA degradation. 00:06:57.05 The gradient is destroyed, because the RNA now can translate anywhere in the cell. To find out more see our. Once it reaches its destination, translation is activated.

00:19:42.26 either in the nucleus or in the cytoplasm for iden... for... for the localization of the RNA 00:16:39.06 And you can see the clock running, down there. 00:28:22.15 And you can see the other cells, the other fibroblasts, moving across the screen to the top, 00:10:48.09 So, that wasn't a very good approach.

00:08:38.06 an open reading frame, so they could be translated. 00:27:44.19 And it will be, then, released from this untranslatable form when it gets to the right place 00:12:38.02 So, in order to prove that this... these events are diffusion, you have to track A patented in situ hybridization technique his lab developed for detecting RNA in morphologically preserved cells revealed that messenger RNA can localize in specific cellular compartments. 00:15:50.13 Now, in order to do this, we had to make a microscope that was able to record at PMCID: PMC4371258 An RNA biosensor for imaging the first round of translation from single cells to living animals.

In vivo imaging of labelled endogenous β-actin mRNA during nucleocytoplasmic transport. 2006 May 23;16(10):R371-3. 00:10:57.22 really one of diffusion. 00:21:58.07 how does the cell know to degrade just those RNAs and not normal RNAs that are necessary Nature 467(7315):604-607. 00:29:04.03 And even the ones that are stationary eventually move. 00:07:21.25 to the right place in the cell. 00:21:38.22 of the cell cycle. 00:16:32.13 So, the center of the pore is right here. 00:10:41.18 You'd expect that the RNA would get translated immediately, but it doesn't. 00:06:21.10 For instance, if that cell develops a chemoresistance during therapy, that cell... 00:21:33.23 have to then get degraded so they don't make proteins and inhibit the progress 00:02:19.27 and these two proteins, Puf6 and Khd1, in the case of the ASH1 message for... for yeast, that goes 00:08:11.24 So, the MS2, here, stem-loop does not see PP7, and vice-versa, so we have two colors. Current Exchange 00:30:47.04 And then an RNA shows up, and a second RNA shows up. 00:07:33.18 Ribosomes loaded up in the periphery region making actin, or in the areas near synaptic spines 00:13:49.08 of where RNA is being born, because that's a fountain of RNA that's... that's being produced there. 00:22:26.12 I think most mRNAs are localized somewhere in the cell; they're not accidentally 00:16:29.07 and where those polymerases are. Click the Policies tab above to see our full Confidentiality & Reporting Policy.

00:26:46.06 And we make a mouse that's got every actin gene tagged with these 1.2 kilobases of stem-loops.

00:03:01.05 which causes this RNA granule to disassociate, releasing RNA for some local translation.

00:11:43.22 which are important in germline development at the posterior Drosophila embryo, are not activated 00:01:31.02 that the RNA has to come out to the... out of the nucleus.

00:21:25.28 because RNAs which are made during the cell cycle for particular portions of the cell cycle 00:33:57.13 And we know one thing about what's required. 00:11:12.15 So, this is a single gene that's actually an integrated virus in a human chromosome 1. The intracellular RNA sorting system: Postal zones, zip codes, mail bags and mail boxes. National Academy of Sciences Robert Singer is a professor at The Albert Einstein College of Medicine in the Departments of Anatomy and Structural Biology, Cell Biology, and Neuroscience, and co-director of the Gruss Lipper Biophotonics Center. Robert H Singer, PhD Harold and Muriel Block Chair Professor & Co-Chair of Anatomy & Structural Biology Professor of Cell Biology Professor of Neuroscience Co-Director, Gruss Lipper Biophotonics Center Co-Director, Integrated Imaging Program robert.singer@einstein.yu.edu Golding Room 601BA, +1.718.430.8646, faculty profile 00:04:29.17 And that makes the RNA molecule very bright and allows you to follow it in time. 00:28:40.11 It goes back and forth, sort of cruising around. 00:18:44.21 And that's because, stochastically, the problem is a ribosome finding an RNA or a polymerase Twitter CSHL Courses 00:20:47.01 to make enough actin molecules to actually change that synaptic structure. He is also a Senior Fellow at the Janelia Research Campus of the Howard Hughes Medical Institute. 00:25:54.24 And that's what we've done through these series of lectures is just watched to see what RNA does, 00:10:14.17 So, originally we went through a series of probe development. 00:06:41.28 And then it goes through the DNA that's coding for the stem-loops. 00:02:45.02 actually, at video rate, 33 frames per second. 00:03:09.09 We can use either this, a northern blot, or we can use a microarray. 00:17:21.21 And one can mix the colors so that some RNAs are blue and green, and some are blue and red, 00:32:41.09 in one direction -- they only go toward the bud -- the myosin is... Following Single Messenger RNA Molecules From Birth To Death In Yeast, National Institute Of General Medical Sciences, National Institute Of Biomedical Imaging And Bioengineering, Light-Activated Gene Expression In Single Cells Within Tissue, Probes For Multiplexing Single RNA Molecule Detectiion In Living Cells, RNA Transport And Localization In Yeast In Situ, Light-Activated Gene Expression In Single Cells, Single RNA Molecule Movement Visualized In Living Cells, High Speed Confocal Photomanipulation Microscopyfor Use In Multi-User Facility, Light-Activated Gene Expression In Single Cells(Rmi), National Institute Of Arthritis And Musculoskeletal And Skin Diseases, Fish &Chips: Single Cell Expression Of Cancer Genes, Light-Activated Gene Expression In Single Cells (RMI), Fish And Chips: Single Cell Expression Of Cancer Genes, Fish And Chips--Single Cell Expression Of Cancer Genes, In Situ Hybridization Visualized--Non-Isotopic Probes, In Situ Hybridization Visualized Using Biotinated Probes, Eunice Kennedy Shriver National Institute Of Child Health &Human Development, In Situ Hybidization Visualized Using Biotinated Probes, Search all U.S. specialist profiles and refer a patient, Read the latest clinical news and earn CME/CEU credits. 00:05:31.20 And you can see in the... in the cells, here, some are yellow, but some are green and some and red, 00:03:32.23 And the coat protein binds the... coats the RNA genome and prevents it from degradation. 00:11:56.19 to its target RNA, whatever the sequence is, and the fluorescence would tell you 00:26:20.08 And this makes a lot of sense, because if you're gonna localize a messenger RNA, Maps & Directions. 00:01:14.21 allow us to see various aspects of mRNA translation and where it occurs in the cell. 00:17:03.04 into the cytoplasm.

00:32:03.20 And diploids are important, as you know, in biology. Doximity / States / Iowa / Waterloo / Robert Singer, MD, Dr. Robert Singer, Dr. Robert Singer, MD, Dr. R Singer, Dr. Robert Harold Singer. 00:34:23.13 And we'll go, in the next talk, into looking at these processes occurring in real time. 00:23:26.13 It can disappear from the cytoplasm within a minute. 00:16:16.06 and the yellows are proteins... 00:21:18.00 very resistant to disintegration, so they maintain their RNA in... in the right places in the cell.